Tissue culture container

Abstract

To provide a novel tissue culture vessel that comprises a plurality of wells for housing and culturing a tissue, and in which replacement of culture solution and tissue recovery after culture are easy.SOLUTION: A tissue culture vessel comprises: an outer vessel 10 which includes a first space 13 storing culture solution L1; a bottom member 20 equipped with a plurality of connected well bottom parts 21 which is separably housed in the first space 13; and a peripheral wall member 30 equipped with a plurality of connected well peripheral wall parts 31 which is separably housed in the first space 13, where the plurality of well bottom parts 21 and the plurality of well peripheral wall parts 31 form a plurality of wells 40 each of which includes a second space 41 for housing and culturing a tissue T, each second space 41 is defined by one of the well bottom parts 21 and one of the well peripheral wall parts 31, and a gap 42 is formed between at least a part of a circumferential direction of a peripheral edge 21a of the well bottom parts 21 and the well peripheral wall parts 31 in each well 40.SELECTED DRAWING: Figure 2

Description

Technical Field

[0001] This specification discloses a tissue culture container.

Background Art

[0002] 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 for inducing the differentiation of pluripotent stem cells into various cells such as cardiomyocytes, retinal pigment epithelial cells, and chondrocytes have been reported.

[0003] For example, Patent Document 1 discloses an intestinal organoid having a structure enclosing a cavity and having a length in the major axis direction of 5 mm or more, which is induced from embryonic stem cells and / or induced pluripotent stem cells. According to Patent Document 1, the intestinal organoid can be used for the development of drugs for preventing or treating intestinal-related diseases and for the pathological study of intestinal-related diseases.

[0004] Patent Document 2 describes a container for culturing a cell mass, which includes a well having a culture space capable of accommodating the cell mass and a culture solution, and a cylindrical body disposed on a surface having an opening of the well and having a lumen communicating with the culture space. One or more communication portions are formed in the cylindrical wall of the cylindrical body to allow the culture solution to be discharged without passing the cell mass outside the cylindrical body. According to Patent Document 2, with this container, the influence on the cell mass is small and the culture solution can be efficiently exchanged.

[0005] Similarly, Patent Document 3 by the same applicant as Patent Document 2 also describes a culture container for a cell mass in which a gap narrower than the cell mass is provided in a cylindrical partition standing at the opening of the well. According to Patent Document 3, with this container, the culture solution can be exchanged without giving damage or stimulation to the cell mass as much as possible, and the productivity of the cell mass can also be improved.

[0006] Patent Document 4 discloses a plate that is immersed in the liquid at a distance from the bottom wall of a container for storing the liquid and holds an object, which can carry the object and has one or more recesses formed in the bottom with through-holes. 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 installed in a container for storing the liquid, the recess can be used as a well for culturing.

Prior Art Documents

Patent Documents

[0007]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Summary of the Invention

Problems to be Solved by the Invention

[0008] When an agent such as a drug candidate substance or a staining substance is allowed to act on a tissue as described in Patent Document 1, it is conceivable to incubate by accommodating the tissue and a culture solution containing the agent in each well of a multi-well plate. At this time, in a normal multi-well plate, since it takes time to replace the culture solution, there is a problem that the treatment time with the culture solution containing the agent differs for each well. Also, since the culture solution cannot be replaced in a short time, there is a problem that the damage to the tissue is large. Further, in order to recover the tissue accommodated in each well after culturing, it is necessary to aspirate and recover the tissue in each well respectively, and there is a problem that the recovery cannot be performed in a short time.

[0009] The containers described in Patent Documents 2 and 3 can be tilted as a whole including the wells to discharge the culture solution in the wells. However, it is considered that complete replacement of the culture solution in the wells is not easy. In addition, the recovery of the tissue after culturing needs to be performed for each well.

[0010] According to the technique described in Patent Document 4, since through holes are formed at the bottom of each recess for holding the object to be cultured, it is considered not suitable for microscopic observation of the object to be cultured. In addition, the recovery of the tissue after culturing needs to be performed for each recess. Therefore, this specification discloses a new tissue culture container that includes a plurality of wells for accommodating and culturing tissues, and enables easy replacement of the culture solution and recovery of the tissue after culturing.

Means for Solving the Problems

[0011] This specification discloses a tissue culture container according to a first embodiment described in any one of the following (1) to (13), and a tissue culture container according to a second embodiment described in any one of the following (14) to (24).

[0012] (1) A tissue culture container for culturing tissues, comprising an outer container bottom and an outer container peripheral wall portion erected from the periphery of the outer container bottom, the outer container including a first space defined by the outer container bottom and the outer container peripheral wall portion, a bottom member having a plurality of connected well bottoms separably accommodated in the first space, a peripheral wall member having a plurality of connected well peripheral wall portions separably accommodated in the first space, and the plurality of well bottoms and the plurality of well peripheral wall portions form a plurality of wells each containing a plurality of second spaces, each second space of each well of the plurality of wells is defined by one of the well bottoms and one of the well peripheral wall portions erected from the periphery of one of the well bottoms, In each of the wells, a gap is formed between at least a part of the circumferential direction of the periphery of the well bottom and the well peripheral wall portion to communicate the second space and the first space in each well. A tissue culture container, characterized in that. (2) The tissue culture container according to (1), wherein the gaps in the respective wells are formed at the same circumferential positions with respect to the well bottoms of the respective wells. (3) The tissue culture container according to (1) or (2), wherein the widths of the gaps in the respective wells are continuously and / or stepwise different depending on the circumferential positions of the peripheries of the well bottoms of the respective wells. (4) The bottom member further includes a first engaging portion. The peripheral wall member further includes a second engaging portion. The outer container includes a third engaging portion that engages with the first engaging portion of the bottom member to position the bottom member on the outer peripheral wall portion of the outer container, and a fourth engaging portion that engages with the second engaging portion of the peripheral wall member to position the peripheral wall member. At least one of the engagement between the first engaging portion of the bottom member and the third engaging portion of the outer container and the 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 be able to change the width of the gap in each well. (1) The tissue culture container according to any one of (1) to (3). (5) At least one of the first engaging portion of the bottom member and the third engaging portion of the outer container is processable so that the position of the bottom member with respect to the peripheral wall member can be adjusted, and / or At least one of the second engaging portion of the peripheral wall member and the fourth engaging portion of the outer container is processable so that the position of the peripheral wall member with respect to the bottom member can be adjusted. (4) The tissue culture container according to (4). (6) The first engaging portion of the bottom member and the third engaging portion of the outer container can engage at a plurality of positions so that the bottom member is positioned at a plurality of different positions with respect 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 engage at a plurality of positions so that the peripheral wall member can be positioned at a plurality of different positions with respect to the bottom member. The tissue culture container according to (4). (7) Two or more of the bottom members are provided. Each of the two or more bottom members includes a first engaging portion that engages with the third engaging portion of the outer container to position the bottom member at a different position with respect to the peripheral wall member. The tissue culture container 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 position of the bottom member with respect to the peripheral wall member by adjusting the interval between the first engaging portion of the bottom member and the third engaging portion of the outer container, and / or A second spacer member that is removably interposed between the second engaging portion of the peripheral wall member and the fourth engaging portion of the outer container, and adjusts the position of the peripheral wall member with respect to the bottom member by adjusting the interval between the second engaging portion of the peripheral wall member and the fourth engaging portion of the outer container. further comprises The tissue culture container according to (4). (9) Two or more of the peripheral wall members are provided. Each of the two or more peripheral wall members includes a second engaging portion that engages with the fourth engaging portion of the outer container to position the peripheral wall member at a different position with respect to the bottom member. The tissue culture container according to (4). (10) The bottom member further includes a first engaging portion. The peripheral wall member further includes a second engaging portion that engages with the first engaging portion of the bottom member to position the peripheral wall member. The outer container includes a third engaging portion that engages with the first engaging portion of the bottom member on the outer container peripheral wall of the outer container to position the bottom member. At least one of the engagement between the first engagement portion of the bottom member and the third engagement portion of the outer container and the engagement between the first engagement portion of the bottom member and the second engagement portion of the peripheral wall member is adjustable so as to be able to change the width of the gap in each well. (1)~(3) The tissue culture container according to any one of (1) to (3). (11) Comprising N peripheral wall members from the first peripheral wall member to the Nth peripheral wall member, The inner diameter of the (n - 1)th well peripheral wall portion, which is each of the plurality of well peripheral wall portions provided by the (n - 1)th peripheral wall member, is larger than the outer diameter of the nth well peripheral wall portion, which is each of the plurality of well peripheral wall portions provided by the nth peripheral wall member. The first peripheral wall member to the Nth peripheral wall member are removably combined so that the nth well peripheral wall portion of the nth peripheral wall member is inserted into the (n - 1)th well peripheral wall portion of the (n - 1)th peripheral wall member. N is an integer of 2 or more, and n is an integer of 2 or more and N or less. Each well is formed by one of the well bottoms and one of the combinations from the first well peripheral wall portion to the Nth well peripheral wall portion. The second space of each well is defined by one of the well bottoms and one of the combinations from the first well peripheral wall portion to the Nth well peripheral wall portion erected from the periphery of one of the well bottoms. In each well, the distance from the lower end of the (n - 1)th well peripheral wall portion to the well bottom is larger than the distance from the lower end of the nth well peripheral wall portion to the well bottom. (1)~(3) The tissue culture container according to any one of (1) to (3). (12) Further comprising a lid that can cover the outer container. The bottom member further comprises a fifth engagement portion. The peripheral wall member further comprises a sixth engagement portion. The lid comprises 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. (1)~(11) The tissue culture container according to any one of (1) to (11). (13) The tissue culture container according to any one of (1) to (12), wherein the bottom of the outer container and the bottom of the well of the bottom member are made of a glass material.

[0013] (14) A tissue culture container for culturing tissues, comprising an outer container bottom and an outer container peripheral wall portion erected from the periphery of the outer container bottom, the outer container enclosing a first space defined by the outer container bottom and the outer container peripheral wall portion, a peripheral wall member including a plurality of connected well peripheral wall portions separably accommodated in the first space, and comprising the outer container bottom and the plurality of well peripheral wall portions form a plurality of wells each enclosing a plurality of second spaces, the second space of each well of the plurality of wells is defined by one of a plurality of portions of the outer container bottom and one of the well peripheral wall portions erected from one of the portions, in each well, a gap is formed between at least a part of the circumferential direction of the periphery of the portion of the outer container bottom and the well peripheral wall portion to communicate the second space in each well with the first space, A tissue culture container, characterized in that. (15) The tissue culture container according to (14), wherein the gap in each well is formed at the same circumferential position with respect to the portion of the outer container bottom of each well. (16) The tissue culture container according to (14) or (15), wherein the width of the gap in each well is continuously and / or stepwise different depending on the circumferential position of the periphery of the portion of the outer container bottom of each well. (17) The peripheral wall member further comprises a second engaging portion, the outer container comprises a fourth engaging portion that engages with the second engaging portion of the peripheral wall member on the outer container peripheral wall portion of the outer container to position the peripheral wall member, at least one of the 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 be able to change the width of the gap in each well. The tissue culture container according to any one of (14) to (16). (18) At least one of the second engaging portion of the peripheral wall member and the fourth engaging portion of the outer container is processable so that the position of the peripheral wall member with respect to the bottom of the outer container can be adjusted. The tissue culture container according to (17). (19) The second engaging portion of the peripheral wall member and the fourth engaging portion of the outer container can engage at a plurality of positions so that the peripheral wall member is positioned at a plurality of different positions with respect to the bottom of the outer container. The tissue culture container according to (17). (20) A second spacer member that is removably interposed between the second engaging portion of the peripheral wall member and the fourth engaging portion of the outer container, and adjusts the position of the peripheral wall member with respect to the bottom of the outer container by adjusting the interval between the second engaging portion of the peripheral wall member and the fourth engaging portion of the outer container. further comprising The tissue culture container according to (17). (21) comprising two or more of the peripheral wall members Each of the two or more peripheral wall members is provided with a second engaging portion that engages with the fourth engaging portion of the outer container to position the peripheral wall member at a different position with respect to the bottom of the outer container. The tissue culture container according to (17). (22) comprising N peripheral wall members from a first peripheral wall member to an Nth peripheral wall member The inner diameter of the (n - 1)th well peripheral wall portion, which is each of the plurality of well peripheral walls provided by the (n - 1)th peripheral wall member, is larger than the outer diameter of the nth well peripheral wall portion, which is each of the plurality of well peripheral walls provided by the nth peripheral wall member, and the first peripheral wall member to the Nth peripheral wall member are removably combined so that the nth well peripheral wall portion of the nth peripheral wall member is inserted into the (n - 1)th well peripheral wall portion of the (n - 1)th peripheral wall member. N is an integer of 2 or more, and n is an integer of 2 or more and N or less. Each well is formed by one of a plurality of portions of the bottom of the outer container and one of the combinations from the first well peripheral wall portion to the Nth well peripheral wall portion. The second space of each well is defined by one of a plurality of portions of the bottom of the outer container and one of the combinations from the first well peripheral wall portion to the Nth well peripheral wall portion erected from the periphery of one of the portions. In each well, the distance from the lower end of the (n - 1)th well peripheral wall portion to the bottom of the outer container is greater than the distance from the lower end of the nth well peripheral wall portion to the bottom of the outer container. The tissue culture container according to any one of (14) to (16). (23) Further comprising a lid body that can cover the outer container. The peripheral wall member further comprises a sixth engaging portion. The lid body comprises an eighth engaging portion that engages with the sixth engaging portion of the peripheral wall member to hold the peripheral wall member. The tissue culture container according to any one of (14) to (22). (24) The tissue culture container according to any one of (14) to (23), wherein the bottom of the outer container is made of a glass material.

Advantages of the Invention

[0014] According to the tissue culture container according to one or more aspects of the present disclosure, a plurality of tissues can be cultured at once, and the replacement of the culture solution and the recovery of the tissue after culture can be performed in a short time.

Brief Description of the Drawings

[0015]

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DETAILED DESCRIPTION OF THE INVENTION

[0016] <Tissue culture container according to the first embodiment> Specific embodiments of the tissue culture container according to the first embodiment of the present disclosure will be described with reference to the drawings.

[0017] FIG. 1 is an exploded perspective view of a tissue culture container according to the first embodiment of the present disclosure. FIGS. 2 to 10 are schematic cross-sectional views showing a method of culturing tissue in a culture solution using the tissue culture container according to the first embodiment shown in FIG. 1. In the following description, with reference to the reference numerals shown in FIGS. 1 to 10, the structure of the tissue culture container 1 according to the first embodiment and its usage method will be described.

[0018] The tissue culture container 1 includes an outer container 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 portion 12 erected from a peripheral edge 11a of the outer container bottom 11, and encloses a first space 13 defined by the outer container bottom 11 and the outer container peripheral wall portion 12. Culture solutions L1, L2, 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 has a periphery defined by the outer container peripheral wall portion 12.

[0019] The bottom member 20 includes a plurality of connected well bottoms 21 that are separably accommodated in the first space 13. The plurality of well bottoms 21 may be connected via a well bottom connection portion 22 as in the illustrated example.

[0020] The peripheral wall member 30 includes a plurality of connected well peripheral wall portions 31 that are separably accommodated in the first space 13. The plurality of well peripheral wall portions 31 may be connected via a well peripheral wall connection portion 32 as in the illustrated example.

[0021] The plurality of well bottoms 21 and the plurality of well peripheral wall portions 31 form a plurality of wells 40, each of which encloses a plurality of second spaces 41. Here, the second space 41 of each of the plurality of wells 40 (each well) is defined by one of the well bottoms 21 and one of the well peripheral wall portions 31 erected from a peripheral edge of one of the well bottoms 21. Each of the second spaces 41 is a space for accommodating and culturing tissue.

[0022] In each well 40, it is preferable that the surface of the well bottom 21 on the side of the second space 41 (the inner bottom surface of the well 40) is a surface (low adhesion surface) subjected to a treatment for suppressing cell adhesion. Also, in each well 40, it is preferable that the surface of the well peripheral wall portion 31 on the side of the second space 41 (the inner side surface of the well 40) is a surface (low adhesion surface) subjected to a treatment for suppressing cell adhesion. The tissue culture vessel 1 in these embodiments is particularly suitable for the purpose of culturing the intestinal organoids or spheroids described later in the second space 41 of each well 40.

[0023] And in each well 40, a gap 42 that communicates the second space 41 and the first space 13 in each well 40 is formed between at least a part of the circumferential direction of the periphery 21a of the well bottom 21 and the well peripheral wall portion 31.

[0024] The well peripheral wall portion 31 is preferably a cylindrical body that is open at both the upper and lower ends. In the illustrated example, the inner diameter of the well peripheral wall portion 31 is uniform regardless of the position in the depth direction of the well 40, but it may have an inner diameter that continuously or stepwise varies depending on the position in the depth direction of the well. For example, in an example not shown, the inner diameter of the well peripheral wall portion 31, which is a cylindrical body, is the smallest at the lower end 31a, continuously expands from the lower end 31a toward the upper end 31b that constitutes the opening of the well 40, and is constant from a position on the way to the upper end 31b to the upper end 31b.

[0025] The culture solutions L1, L2, and L3 stored in the first space 13 of the outer container 10 can flow through the gaps 42 into the second space 41 of each of the plurality of wells 40 and fill the second space 41. Further, when the culture solutions 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 solutions L1, L2, and L3, the culture solutions L1, L2, and L3 in the second space 41 of each of the plurality of wells 40 can be simultaneously discharged through the gaps 42. Also, in the tissue culture container 1 including the plurality of wells 40 in which the second space 41 is filled with the culture solutions L1, L2, and L3 in the outer container 10, even when the bottom member 20 and the peripheral wall member 30 are separated from the outer container 10 in a combined state, the culture solutions L1, L2, and L3 in the second space 41 of each of the plurality of wells 40 can be discharged through the gaps 42. That is, according to the tissue culture container 1, it is possible to exchange the culture solutions L1, L2, and L3 in the plurality of wells 40 each containing the tissue T at once in a short time. Also, it is possible to treat the tissues T accommodated in each of the plurality of wells 40 with the culture solutions L1, L2, and L3 under uniform conditions.

[0026] Furthermore, when the bottom member 20 and the peripheral wall member 30 are separated from the outer container 10 after the treatment with the culture solutions L1, L2, and L3 using the tissue culture container 1 is completed, the wells 40 are disassembled and the plurality of tissues T are released into the first space 13 of the outer container 10, so that the plurality of tissues T can be collected at once.

[0027] The gap 42 in the well 40 is formed between at least a part of the circumferential direction of the peripheral edge 21a of the well bottom 21 and the well peripheral wall portion 31. The gap 42 may have a width through which the tissue T to be cultured cannot pass. For example, the width of the gap 42 is preferably 50% or less of the width of the tissue T to be cultured.

[0028] In the tissue culture container 1 according to the example shown in FIGS. 1 to 10, the gap 42 in the well 40 is formed between the peripheral edge 21a of the well bottom 21 and the lower end 31a of the well peripheral wall portion 31 over the entire circumferential direction of the peripheral edge 21a of the well bottom 21, and its width is uniform. According to this configuration, the following effects are achieved. That is, in the tissue culture container 1 in which the outer container 10, the bottom member 20, and the peripheral wall member 30 are combined and the first space 13 and the second space 41 are filled with the culture solutions L1, L2, and L3, the culture solutions L1, L2, and L3 can be discharged from the first space 13 and the second space 41 regardless of the direction in which the whole is tilted.

[0029] Further, it is preferable that the gaps 42 in each of the plurality of wells 40 are formed at the same circumferential position with respect to the well bottom 21 of each well 40 in a plan view of the tissue culture container 1. In this case, the gap 42 in the well 40 may be formed only in a part of the circumferential direction of the peripheral edge 21a of the well bottom 21. If the whole of the tissue culture container 1 is tilted so that the position of the gap 42 in each well 40 is downward, the culture solutions L1, L2, and L3 can be discharged from the first space 13 and the second space 41.

[0030] Also, the width of the gap 42 in each of the plurality of wells 40 may be uniform over the entire circumferential direction of the peripheral edge 21a of the well bottom 21, as in the tissue culture container 1 of the example shown in FIGS. 1 to 10, or may be continuously and / or stepwise different depending on the circumferential position of the peripheral edge 21a of the well bottom 21. According to the tissue culture container 1 in which the width of the gap 42 in each of the plurality of wells 40 is different depending on the circumferential position of the peripheral edge 21a of the well bottom 21, when the whole of the tissue culture container 1 is tilted to discharge the culture solutions L1, L2, and L3 from the first space 13 and the second space 41, it is possible to change the discharge rate of the culture solutions L1, L2, and L3 from the second space 41 by changing the tilting direction.

[0031] Fig. 11 shows an example of the tissue culture container 1 having the same features as those in Figs. 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 continuously varies depending on the circumferential position of the periphery 21a of the well bottom 21. In the example shown in Fig. 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 continuously varies depending on the circumferential position of the periphery 21a of the well bottom 21. The most protruding portion of the convex portion 21b contacts the lower end 31a of the well peripheral wall portion 31, and no gap 42 exists at this portion. 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.

[0032] As used herein, the "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 in each well 40 of the tissue culture container 1 according to the example shown in Figs. 1 to 10 is each a single continuous opening. However, different from the illustrated embodiment, a gap including a plurality of adjacent openings is also an example of the "gap" herein. As a gap composed of a plurality of adjacent openings, a gap between the well bottom and the well peripheral wall portion in which a member having a plurality of adjacent openings such as a mesh or a fence is arranged so as to partition the first space and the second space can be exemplified.

[0033] The culture solutions L1, L2, and L3 stored in the first space 13 of the outer container 10 can flow through the gap 42 into the second space 41 of each of the plurality of wells 40 and fill the second space 41. Further, when the culture solutions 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 solutions L1, L2, and L3, the culture solutions L1, L2, and L3 in the second space 41 of each of the plurality of wells 40 can be simultaneously discharged through the gap 42. Also, in the tissue culture container 1 including the plurality of wells 40 in which the second space 41 is filled with the culture solutions L1, L2, and L3 in the outer container 10, even when the bottom member 20 and the peripheral wall member 30 are separated from the outer container 10 in a combined state, the culture solutions L1, L2, and L3 in the second space 41 of each of the plurality of wells 40 can be discharged through the gap 42.

[0034] The bottom member 20 further includes a first engaging portion 23. On the other hand, the outer container 10 includes a third engaging portion 14 that engages with the first engaging portion 23 of the bottom member 20 at the upper end portion 12a of the outer container peripheral wall portion 12 to position the bottom member 20. The first engaging portion 23 is preferably a plurality of arm-shaped engaging portions that project from a part of the plurality of well bottoms 21 connected via the well bottom connecting portion 22 toward the third engaging portion 14 of the outer container peripheral wall portion 12 as in the illustrated example. The third engaging portion 14 is preferably an engaging 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.

[0035] The peripheral wall member 30 further includes a second engaging portion 33. On the other hand, the outer container 10 includes a fourth engaging portion 15 that engages with the second engaging portion 33 of the peripheral wall member 30 at the upper end portion 12a of the outer container peripheral wall portion 12 to position the peripheral wall member 30. The second engaging portion 33 is preferably a plurality of arm-shaped engaging portions that project from a part of the plurality of well peripheral wall portions 31 connected via the well peripheral wall connecting portion 32 toward the fourth engaging portion 15 of the outer container peripheral wall portion 12 as in the illustrated example. The fourth engaging portion 15 is preferably an engaging 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.

[0036] As shown in Fig. 2, in the first space 13 of the outer container 10, the bottom member 20 is positioned and accommodated by the engagement between the third engaging portion 14 of the outer container 10 and the first engaging portion 23 of the bottom member 20. Further, in the first space 13 of the outer container 10, the peripheral wall member 30 is positioned and accommodated by the engagement between the fourth engaging portion 15 of the outer container 10 and the second engaging portion 33 of the peripheral wall member 30. As a result, a plurality of wells 40 with a gap 42 formed therebetween are formed. The shapes of and the mutual engagement states between the first engaging portion 23 of the bottom member 20, the second engaging portion 33 of the peripheral wall member 30, the third engaging portion 14 and the fourth engaging 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 gap 42.

[0037] As shown in Fig. 2, using the tissue culture container 1 in which the outer container 10, the bottom member 20 and the peripheral wall member 30 are combined, tissues T are respectively accommodated and cultured in a plurality of wells 40. When observing the tissues T being cultured in the plurality of wells 40 from above with a microscope, if the well bottoms 21 of the respective wells 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 bottoms 21 of the respective wells 40 and the outer container bottom 11 of the outer container 10 are made of a glass material having no autofluorescence. Subsequently, with reference to Figs. 2 to 10, a procedure for treating intestinal organoids as the tissue T using the tissue culture container 1 according to the first embodiment will be described.

[0038] As the intestinal organoid, typically, it is the intestinal organoid described in Patent Document 1. More specifically, it is an intestinal organoid derived from embryonic stem cells and / or induced pluripotent stem cells, having a structure enclosing a cavity, and characterized in that the length in the major axis direction is 5 mm or more. The intestinal organoid described in Patent Document 1 is more preferably composed of endodermal cells, ectodermal cells, and mesodermal cells, and includes intestinal epithelial cells on the outer surface. The intestinal organoid containing endodermal cells, ectodermal cells, and mesodermal cells and including intestinal epithelial cells on the outer surface can absorb substances outside into the cavity through the intestinal epithelial cells on the outer surface. Further, in the intestinal organoid described in Patent Document 1, when the intestinal epithelial cells on the outer surface are further transporter-positive, it is more preferable because substances can be taken up through the transporter. That is, the intestinal organoid containing transporter-positive intestinal epithelial cells has a substance absorption ability similar to that of the intestine. The intestinal organoid more preferably shows the development of microvilli and crypts on the outer surface.

[0039] While the intestinal organoid having the above characteristics has the ability to actively take up substances from the outside into the lumen, it preferably has the ability (barrier property) to prevent the entry of substances that are not the targets of active uptake into the lumen. If the cell layer surrounding the lumen is not sufficiently developed and the barrier property is insufficient due to reasons such as the formation of openings in the cell layer, the intestinal organoid non-specifically absorbs substances that are not the targets of active uptake into the lumen. Therefore, in this specification, a method for discriminating between an intestinal organoid with insufficient barrier property and a normal intestinal organoid with barrier property among a plurality of intestinal organoids is disclosed.

[0040] The method for discriminating between an intestinal organoid with insufficient barrier property and a normal intestinal organoid preferably includes Step 1 of accommodating a test intestinal organoid together with a first culture solution in each well of the plurality of wells in a substrate containing a plurality of wells, After Step 1, replacing the first culture solution in each well with a second culture solution containing a dye and culturing the test intestinal organoid in the second culture solution (Step 2), After step 2, replace the second culture solution in each well with a third culture solution, and wash the test intestinal organoids in the third culture solution in step 3, and After step 3, discriminate the test intestinal organoids stained with the dye from intestinal organoids with insufficient barrier properties, and discriminate the test intestinal organoids not stained with the dye from normal intestinal organoids in step 4 include.

[0041] Step 4 preferably includes at least one of step 4-1 of selectively removing the test intestinal organoids stained with the dye and step 4-2 of selectively collecting only the test intestinal organoids not stained with the dye, and more preferably includes both step 4-1 and step 4-2. As the dye, a water-soluble dye that causes absorption and emission of visible light or ultraviolet light and has no cytotoxicity can be used. Specific examples of the dye include pH indicators that exhibit color near pH 7.4, such as bromophenol red, bromothymol blue, neutral red, and phenolphthalein, and biological dyes such as flavonoids and anthocyanins.

[0042] The method for discriminating intestinal organoids with insufficient barrier properties and normal intestinal organoids, including steps 1 to 4, preferably uses the tissue culture container 1 according to the first embodiment shown in FIG. 1 as the "substrate including a plurality of wells". An example of this method will be described with reference to FIGS. 2 to 10.

[0043] First, as shown in FIG. 2, place one test intestinal organoid T in each of the second spaces 41 of the plurality of wells 40 of the tissue culture container 1 formed by combining the outer container 10, the bottom member 20, and the peripheral wall member 30. At this time, the first culture solution L1 is stored in the first space 13 of the outer container 10, and the first culture solution L1 passes through the gap 42 and fills the second space 41 included in each of the plurality of wells 40. The above is step 1.

[0044] Subsequently, as shown in Fig. 3, while maintaining the state in which a plurality of wells 40 containing the test intestinal organoids T are formed, the bottom member 20 and the peripheral wall member 30 are lifted from the outer container 10. The first culture solution L1 in the second space 41 of the plurality of wells 40 is discharged into the outer container 10 through the gap 42. The first culture solution L1 in the outer container 10 is removed. Subsequently, as shown in Fig. 4, a second culture solution L2 containing a dye is stored in the first space 13 of the outer container 10.

[0045] Subsequently, as shown in Fig. 5, a combination of the bottom member 20 and the peripheral wall member 30 in which a plurality of wells 40 containing the test intestinal organoids T are formed is reinstalled in the outer container 10 storing the second culture solution L2 containing the dye, and the test intestinal 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.

[0046] Instead of the step 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 as shown in Figs. 3 to 4, while maintaining the positional relationship between the outer container 10, the bottom member 20, and the peripheral wall member 30 shown in Fig. 2, the first culture solution L1 is removed from the first space 13 of the outer container 10, and subsequently, the second culture solution L2 is stored in the first space 13 of the outer container 10, and it is also possible to shift from the stage shown in Fig. 2 to the stage shown in Fig. 5.

[0047] The culture time (staining time) in step 2 can be appropriately adjusted according to the type and concentration of the dye. For example, when phenol red is used as the dye, about 3 hours can be exemplified. After step 2, unstained test intestinal organoids T1 and stained test intestinal organoids T2 are produced by the dye.

[0048] After culturing in step 2, as shown in Fig. 6, while maintaining the state in which a plurality of wells 40 containing the test intestinal organoids T1 and T2 are formed, the bottom member 20 and the peripheral wall member 30 are lifted from the outer container 10. The second culture solution L2 in the second space 41 of the plurality of wells 40 is discharged into the outer container 10 through the gap 42. Subsequently, as shown in FIG. 7, the second culture solution L2 in the outer container 10 is removed.

[0049] Subsequently, as shown in FIG. 8, the bottom member 20 and the peripheral wall member 30 in a state where a plurality of wells 40 containing the test intestinal organoids T1 and T2 are formed are reinstalled in the outer container 10 storing the third culture solution L3, and the test intestinal organoids T1 and T2 are washed in the third culture solution L3. This step is step 3. The third culture solution L3 may be any culture solution that can wash the pigment adhering to the surfaces of the test intestinal organoids T1 and T2, and preferably, it is a colorless and transparent culture solution.

[0050] Instead of the step 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 as shown in FIGS. 6 to 7, while maintaining the positional relationship between the outer container 10, the bottom member 20, and the peripheral wall member 30 shown in FIG. 5, the second culture solution L2 is removed from the first space 13 of the outer container 10, and subsequently, the third culture solution L3 is stored in the first space 13 of the outer container 10, and it is also possible to shift from the stage shown in FIG. 5 to the stage shown in FIG. 8.

[0051] After step 3, the test intestinal organoid T2 stained with the pigment is discriminated as an intestinal organoid with insufficient barrier properties, and the test intestinal organoid T1 not stained with the pigment is discriminated as a normal intestinal organoid. This step is step 4.

[0052] Step 4 preferably includes, as shown in FIG. 9, selectively removing the test intestinal organoid T2 stained with the pigment from the well 40 containing it. This step is a specific example of step 4-1.

[0053] Step 4 further includes, after removing the test intestinal organoid T2 by step 4-1, as shown in FIG. 10, removing the peripheral wall member 30 and collecting the normal test intestinal organoids T1 not stained with the pigment together in the first space 13 of the outer container 10. This step is a specific example of step 4-2.

[0054] Thus, it is advantageous to carry out a method for discriminating between intestinal organoids with insufficient barrier properties, including steps 1 to 4, and normal intestinal organoids, using the tissue culture vessel 1 in the following respects.

[0055] First, compared with the case of replacing the medium in each well using a normal multi-well plate, the replacement from the first culture solution L1 to the second culture solution L2 and the replacement from the second culture solution L2 to the third culture solution L3 can be carried out in a short time. Also, the replacement of the medium can be completed for all wells at substantially the same timing. For this reason, it becomes possible to perform a staining treatment on a plurality of test intestinal organoids T accommodated in each of the plurality of wells 40 under gentle and uniform conditions.

[0056] Second, normal test intestinal organoids T1 that are not stained with a dye and are accommodated in each of the plurality of wells 41 can be collectively recovered into the first space 13 of the outer container 10 by removing the peripheral wall member 30 from the outer container 10 (and in some cases further removing the bottom member 20) and disassembling the plurality of wells 41. When recovering normal test intestinal organoids T1 in each well using a normal multi-well plate, it is necessary to individually recover the test intestinal organoids T1 in each well, making it difficult to recover them in a short time and there is a possibility of damaging the test intestinal organoids T1. However, these problems can be solved by using the tissue culture vessel 1.

[0057] Since the tissue culture vessel 1 according to the present disclosure can quickly replace the culture solution, it is particularly useful in a method for treating a plurality of tissues T under the same conditions at once in a short time. Phenol red is usually used as a pH indicator. Since a culture solution containing phenol red easily changes color when it comes into contact with air, in the above method for discriminating between intestinal organoids with insufficient barrier properties, including steps 1 to 4, and normal intestinal organoids, when using phenol red as a dye, it is necessary to treat a plurality of intestinal organoids T with a culture solution containing phenol red under short-time and uniform conditions. The tissue culture vessel 1 according to the present disclosure is particularly suitable for such applications.

[0058] In addition to the method for discriminating between non-barrier intestinal organoids and normal intestinal organoids, which includes steps 1 to 4, the tissue culture container 1 according to the present disclosure can be used for various applications that take advantage of the ability to perform a complete exchange of the total amount of the culture medium in a plurality of wells in a short time. For example, the tissue culture container 1 can be used in tests for finely verifying the process by which tissue T is affected by a drug or the like, and tests using compounds that are easily affected by pH or light. The tissue culture container 1 can also be used in various methods for treating a plurality of tissues T under the same conditions at once. For example, the tissue culture container 1 can be used in a method for determining a tissue T that is resistant or sensitive to a poison by treating a plurality of tissues T with a culture solution containing the poison, or in a method for discriminating a tissue T that has the ability to absorb a substance by treating a plurality of tissues T with a culture solution containing the substance to be absorbed.

[0059] <Modification Example 1 of the First Embodiment> As a modification example 1 of the tissue culture container 1 according to the first embodiment of the present disclosure, there is provided a tissue culture container 1 further including a lid body 50 shown in FIGS. 12 and 13.

[0060] In the tissue culture container 1 of modification example 1 of the first embodiment, the lid body 50 covers the opening of the first space 13 of the outer container 10 and is separable from the outer container 10.

[0061] The tissue culture container 1 of modification example 1 of the first embodiment further includes the bottom member 20 further includes a fifth engaging portion 24, the peripheral wall member 30 further includes a sixth engaging portion 34, the lid body 50 includes a seventh engaging portion 51 that engages with the fifth engaging portion 24 of the bottom member 20 to hold the bottom member 20, and an eighth engaging portion 52 that engages with the sixth engaging portion 34 of the peripheral wall member 30 to hold the peripheral wall member 30.

[0062] In the tissue culture container 1 of modification example 1 of the first embodiment shown in FIGS. 12 and 13, the fifth engaging portion 24 of the bottom member 20 is an engaging claw provided on the tip side of the first engaging portion 23 and protruding toward the seventh engaging portion 51 of the lid body 50.

[0063] In the tissue culture container 1 of Modification 1 of the first embodiment shown in FIGS. 12 and 13, the sixth engaging portion 34 of the peripheral wall member 30 is provided on the tip side of the second engaging portion 33 and is an engaging claw protruding toward the eighth engaging portion 52 of the lid body 50.

[0064] As shown in FIG. 12, in each of the second spaces 41 of the plurality of wells 40 of the tissue culture container 1 of Modification 1 of the first embodiment in which the outer container 10, the bottom member 20, the peripheral wall member 30, and the lid body 50 are combined, one tissue T is accommodated one by one. At this time, the culture solution L is stored in the first space 13 of the outer container 10, and the culture solution L passes through the gap 42 and also fills the second space 41 included in each of the plurality of wells 40.

[0065] In the tissue culture container 1 of Modification 1 of the first embodiment containing the culture solution L and the tissue T in the state shown in FIG. 12, when the culture solution L is exchanged for another culture solution, as shown in FIG. 13, the lid body 50 is lifted from the outer container 10. At this time, due to the engagement between the fifth engaging portion 24 of the bottom member 20 and the seventh engaging portion 51 of the lid body 50, and the engagement between the sixth engaging portion 34 of the peripheral wall member 30 and the eighth engaging portion 52 of the lid body 50, the bottom member 20 and the peripheral wall member 30 are lifted from the outer container 10 while maintaining the state of forming the plurality of wells 40 containing the tissue T together with the lid body 50. The culture solution L in the second space 41 of the plurality of wells 40 is discharged into the outer container 10 through the gap 42. From the state shown in FIG. 13, the culture solution L in the first space 13 of the outer container 10 is exchanged for another culture solution, and then, together with the lid body 50, the combination of the bottom member 20 and the peripheral wall member 30 forming the plurality of wells 40 containing the tissue T is reinstalled in the outer container 10, so that the tissue T can be immersed in a new culture solution.

[0066] Thus, according to the tissue culture container 1 of Modification 1 of the first embodiment, it is particularly easy to remove the bottom member 20 and the peripheral wall member 30 integrally with the lid body 50 from the outer container 10 and reinstall them in the outer container 10, and the exchange of the culture solution can be performed in a short time.

[0067] <Modification 2 of the First Embodiment> As a second modification example of the tissue culture container 1 according to the first embodiment of the present disclosure, 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 can be adjusted so as to change the width of the gap 42 in each well 40. According to the tissue culture container 1 of the second modification example of the first embodiment, the width of the gap 42 in each of the plurality of wells 40 can be changed. The width of the gap 42 suitable for the size of the tissue T to be cultured can be selected.

[0068] A first example of the tissue culture container 1 of the second modification example of the first embodiment is 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 is processable so that the position of the bottom member 20 with respect 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 is processable so that the position of the peripheral wall member 30 with respect to the bottom member 20 can be adjusted, which is the tissue culture container 1.

[0069] Here, "processable" 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.

[0070] In the first example of the tissue culture container 1 of the second modification example of the first embodiment, the engagement position is changed and the gap 42 is adjusted by processing 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.

[0071] A second example of the tissue culture container 1 of the second modification example of the first embodiment is The first engaging portion 23 of the bottom member 20 and the third engaging portion 14 of the outer container 10 can engage at a plurality of positions such that the bottom member 20 is positioned at a plurality of different positions with respect to the peripheral wall member 30. And / or, The second engaging portion 33 of the peripheral wall member 30 and the fourth engaging portion 15 of the outer container 10 can engage at a plurality of positions such that the peripheral wall member 30 is positioned at a plurality of different positions with respect to the bottom member 20. The tissue culture container 1 is such that.

[0072] According to the second example of the tissue culture container 1 of the second modification of the first embodiment, the width of the gap 42 can be appropriately adjusted as the dimensions change due to the growth of the tissue T.

[0073] In the second example of the tissue culture container 1 of the second modification of the first embodiment, the first engaging portion 23 of the bottom member 20, the third engaging portion 14 of the outer container 10, the second engaging portion 33 of the peripheral wall member 30, or the fourth engaging portion 15 of the outer container 10 can be a multi-stage protrusion or recess.

[0074] The third example of the tissue culture container 1 of the second modification of the first embodiment is provided with two or more bottom members 20, Each of the two or more bottom members 20 is a tissue culture container 1 that engages with the third engaging portion 14 of the outer container 10 and is provided with a first engaging portion 23 that positions the bottom member 20 at different positions with respect to the peripheral wall member 30.

[0075] According to the third example of the tissue culture container 1 of the second modification of the first embodiment, an appropriate one can be selected from among the two or more bottom members 20 so that a gap 42 having an appropriate width is formed according to the dimensions of the tissue T to be cultured.

[0076] The fourth example of the tissue culture container 1 of the second modification of the first embodiment is A first spacer member that is removably interposed between the first engaging portion 23 of the bottom member 20 and the third engaging portion 14 of the outer container 10 and adjusts the position of the bottom member 20 with respect to the peripheral wall member 30 by adjusting the interval between the first engaging portion 23 of the bottom member 20 and the third engaging portion 14 of the outer container 10, and / or A second spacer member 60 that is removably interposed between a second engaging portion 33 of the peripheral wall member 30 and a fourth engaging 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 interval between the second engaging portion 33 of the peripheral wall member 30 and the fourth engaging portion 15 of the outer container 10 further includes a tissue culture container 1

[0077] The tissue culture container 1 according to the fourth example of the modification 2 of the first embodiment including the second spacer member 60 is shown in FIGS. 15 and 16. 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 container 1 in a state where the second engaging portion 33 of the peripheral wall member 30 and the fourth engaging portion 15 of the outer container 10 shown in FIG. 15 are directly engaged, the gap 42 in the well 40 is narrow. As shown in FIG. 16, by inserting and interposing the tip portion 61 of the second spacer member 60 between the second engaging portion 33 of the peripheral wall member 30 and the fourth engaging portion 15 of the outer container 10, the interval between the second engaging portion 33 of the peripheral wall member 30 and the fourth engaging portion 15 of the outer container 10 expands, and according to the expansion of the interval, the entire peripheral wall member 30 is lifted upward, and the distance between the peripheral edge 21a of the well bottom 21 and the lower end 31a of the well peripheral wall portion 31, that is, the width of the gap 42 in the well 40 expands. By adjusting the insertion depth of the tip portion 61 of the second spacer member 60 between the second engaging portion 33 of the peripheral wall member 30 and the fourth engaging 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 container 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 becomes possible. First, as shown in FIG. 15, without using the second spacer member 60, the outer container 10, the bottom member 20, and the peripheral wall member 30 are combined so that the second engaging portion 33 of the peripheral wall member 30 and the fourth engaging portion 15 of the outer container 10 are directly engaged, and a tissue culture container 1 having a plurality of wells 40 with a gap 42 smaller than or equal to the size of the cells C (for example, 10 μm or less) is formed. Then, the first space 13 and the second space 41 of the tissue culture container 1 are filled with the culture solution L necessary for spheroid culture, and the cells C are seeded in the second space 41 in each well 40. Culturing is performed in this state to prepare spheroids T from the cells C. As shown in FIG. 16, by inserting and interposing the tip portion 61 of the second spacer member 60 between the second engaging portion 33 of the peripheral wall member 30 and the fourth engaging portion 15 of the outer container 10, the width of the gap 42 in the well 40 is widened. The width of the gap 42 after the intervention of the second spacer member 60 is adjusted to a width (for example, 450 μm or less) through which spheroids and cells C that have not formed spheroids smaller than the desired size can pass, but spheroid tissues T that have grown to a size equal to or greater than the desired size cannot pass. As a result, the spheroid T remains in the well 40 containing the spheroid T that has grown to a size equal to or greater than the desired size, and the spheroids smaller than the desired size and the cells C that have not formed spheroids pass through the gap 42 and are discharged into the first space 13 of the outer container 10 from the other wells 40. Subsequently, in the same manner as in the examples of FIGS. 6 and 7, the bottom member 20 and the peripheral wall member 30 are lifted from the outer container 10 while maintaining the state in which a plurality of wells 40 containing the spheroid T are formed. Subsequently, although not shown, the culture solution L containing spheroids and cells C smaller than the desired size in the outer container 10 is discarded and replaced with another liquid (for example, a cryopreservation solution) as necessary. Thereafter, the combination of the bottom member 20 and the peripheral wall member 30 in the state in which a plurality of wells 40 containing the spheroid T are formed is returned to the outer container 10 containing the other liquid. 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 having a size equal to or greater than the desired size can be recovered into the outer container 10 containing the other liquid.

[0078] The fifth example of the tissue culture container 1 of Modification 2 of the first embodiment is provided with two or more peripheral wall members 30, and the two or more peripheral wall members 30 are each engaged with the fourth engaging portion 15 of the outer container 10 and provided with a second engaging portion 33 for positioning the peripheral wall member 30 at a different position with respect to the bottom member 20, which is the tissue culture container 1.

[0079] According to the fourth example of the tissue culture container 1 of Modification 2 of the first embodiment, an appropriate one can be selected from among the two or more peripheral wall members 30 so that a gap 42 having an appropriate width is formed according to the size of the tissue T to be cultured.

[0080] <Modification 3 of the First Embodiment> As a modification 3 of the tissue culture container 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 position the peripheral wall member 30 with respect to the bottom member 20, and examples include those in which at least one of the engagement between the first engaging portion 23 of the bottom member 20 and the third engaging portion 14 of the outer container 10 and the engagement between the first engaging portion 23 of the bottom member 20 and the second engaging portion 33 of the peripheral wall member 30 can be adjusted so as to change the width of the gap 42 in each well 40. According to the tissue culture container 1 of Modification 3 of the first embodiment, the width of the gap 42 in each of the plurality of wells 40 can be changed. It is possible to select the width of the gap 42 suitable according to the size of the tissue T to be cultured.

[0081] The first example of the tissue culture container 1 of Modification 3 of the first embodiment is at least one of the first engaging portion 23 of the bottom member 20 and the third engaging portion 14 of the outer container 10 is processable so that the position of the bottom member 20 with respect to the peripheral wall member 30 can be adjusted, and / or at least one of the first engaging portion 23 of the bottom member 20 and the second engaging portion 33 of the peripheral wall member 30 is processable so that the position of the peripheral wall member 30 with respect to the bottom member 20 can be adjusted, It is the tissue culture container 1.

[0082] Here, "processable" means that the first engaging portion 23 of the bottom member 20, the third engaging portion 14 of the outer container 10, or the second engaging 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.

[0083] In the first example of the tissue culture container 1 of Modification Example 3 of the first embodiment, by processing the first engaging portion 23 of the bottom member 20, the third engaging portion 14 of the outer container 10, or the second engaging portion 33 of the peripheral wall member 30, the engaging position is changed and the gap 42 is adjusted.

[0084] The second example of the tissue culture container 1 of Modification Example 3 of the first embodiment is The first engaging portion 23 of the bottom member 20 and the third engaging portion 14 of the outer container 10 can engage at a plurality of positions so that the bottom member 20 is positioned at a plurality of different positions with respect to the peripheral wall member 30, and / or The first engaging portion 23 of the bottom member 20 and the second engaging portion 33 of the peripheral wall member 30 can engage at a plurality of positions so that the peripheral wall member 30 is positioned at a plurality of different positions with respect to the bottom member 20, which is the tissue culture container 1.

[0085] According to the second example of the tissue culture container 1 of Modification Example 3 of the first embodiment, the width of the gap 42 can be appropriately adjusted as the dimensions change due to the growth of the tissue T.

[0086] In the second example of the tissue culture container 1 of Modification Example 3 of the first embodiment, the first engaging portion 23 of the bottom member 20, the third engaging portion 14 of the outer container 10, or the second engaging portion 33 of the peripheral wall member 30 can be multi-stage protrusions or recesses.

[0087] The third example of the tissue culture container 1 of Modification Example 3 of the first embodiment is provided with two or more bottom members 20, The tissue culture vessel 1 has two or more bottom members 20, each having a first engaging portion 23 that engages with the third engaging portion 14 of the outer container 10 to position the bottom member 20 at different positions with respect to the peripheral wall member 30.

[0088] According to the third example of the tissue culture vessel 1 of Modification 3 of the first embodiment, an appropriate one can be selected from among two or more bottom members 20 so that a gap 42 having an appropriate width is formed according to the size of the tissue T to be cultured.

[0089] The fourth example of the tissue culture vessel 1 of Modification 3 of the first embodiment is provided with two or more peripheral wall members 30, The two or more peripheral wall members 30 each have a second engaging portion 33 that engages with the first engaging portion 23 of the bottom member 20 to position the peripheral wall member 30 at different positions with respect to the bottom member 20, and is a tissue culture vessel 1.

[0090] According to the fourth example of the tissue culture vessel 1 of Modification 3 of the first embodiment, an appropriate one can be selected from among two or more peripheral wall members 30 so that a gap 42 having an appropriate width is formed according to the size of the tissue T to be cultured.

[0091] <Modification 4 of the First Embodiment> As a modification 4 of the tissue culture vessel 1 according to the first embodiment of the present disclosure, as shown in FIGS. 17 and 18, it includes two peripheral wall members 30, a first peripheral wall member 30-1 and a second peripheral wall member 30-2, The inner diameter of each first well peripheral wall portion 31-1, which is each of the plurality of well peripheral wall portions provided in the first peripheral wall member 30-1, is larger than the outer diameter of each second well peripheral wall portion 31-2, which is each of the plurality of well peripheral wall portions provided in the second peripheral wall member 30-2. The first peripheral wall member 30-1 and the second peripheral wall member 30-2 are removably combined 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. 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 combination of the first well peripheral wall portion 31-1 and the second well peripheral wall portion 31-2 erected 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 the tissue culture container 1 can be cited.

[0092] The function of the tissue culture container 1 according to this modification 4 will be described with reference to FIGS. 17 and 18. The tissue culture container 1 shown in FIG. 17 has the first peripheral wall member 30-1 and the second peripheral wall member 30-2. In the state where 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 and they are overlapped, the width of the gap 42 of each well 40 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, so it is relatively small. When only the second peripheral wall member 30-2 is removed from the tissue culture container 1 shown in FIG. 17, as shown in FIG. 18, the width of the gap 42 of each well 40 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, so it is relatively large. That is, by starting tissue culture with the tissue culture container 1 according to this modification 4 in a state where all the peripheral wall members composed of the first peripheral wall member 30-1 and the second peripheral wall member 30-2 are overlapped, and removing the second peripheral wall member 30-2 at the stage when the tissue has grown and continuing the culture, it becomes possible to perform tissue culture while gradually widening the width of the gap 42 of 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 for example, 2 to 5 can be exemplified.

[0093] By using the tissue culture container 1 according to Modification Example 4 of the first embodiment, for example, the following spheroid culture becomes possible. First, as shown in FIG. 17, the peripheral wall member 30 formed by combining the first peripheral wall member 30-1 and the second peripheral wall member 30-2, the outer container 10, and the bottom member 20 are combined to form a tissue culture container 1 having a plurality of wells 40 with a gap 42 equal to or smaller than the size of the cell C (for example, 10 μm or smaller). Then, the first space 13 and the second space 41 of the tissue culture container 1 are filled with the culture solution L necessary for spheroid culture, and the cells C are seeded in the second space 41 in each well 40. Culturing is performed in this state to prepare spheroids T from the cells C. Subsequently, as shown in FIG. 18, by removing the second peripheral wall member 30-2, the width of the gap 42 in the well 40 is widened. The width of the gap 42 after removing the second peripheral wall member 30-2 is adjusted to a width (for example, 450 μm or smaller) through which the spheroid tissue T that has grown to a desired size or more does not pass, but the spheroids smaller than the desired size and the cells C that have not formed spheroids pass. As a result, the spheroids T remain in the wells 40 containing the spheroids T that have grown to a desired size or more, and the spheroids smaller than the desired size and the cells C that have not formed spheroids pass through the gap 42 and are discharged into the first space 13 of the outer container 10 from the other wells 40. Subsequently, as in the examples of FIGS. 6 and 7, the bottom member 20 and the peripheral wall member 30 are lifted from the outer container 10 while maintaining the state in which a plurality of wells 40 containing the spheroids T are formed. Subsequently, although not shown, the culture solution L containing the spheroids and the cells C smaller than the desired size in the outer container 10 is discarded and replaced with another liquid (for example, a cryopreservation solution) as necessary. Thereafter, the combination of the bottom member 20 and the peripheral wall member 30 in the state in which a plurality of wells 40 containing the spheroids T are formed is returned to the outer container 10 containing the other liquid. 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 having a desired size or more can be recovered into the outer container 10 containing the other liquid.

[0094] <Tissue culture container according to the second embodiment> A specific aspect of the tissue culture container according to the second embodiment of the present disclosure will be described with reference to FIG. 14. In the tissue culture container according to the second embodiment, the components having the same features and functions as those of the tissue culture container according to the first embodiment are denoted by the same reference numerals, and the description thereof will be omitted.

[0095] FIG. 14(A) is a schematic plan view of a tissue culture container 1 according to the second embodiment of the present disclosure. FIG. 14(B) is a schematic cross-sectional view of the tissue culture container 1 according to the second embodiment of the present disclosure.

[0096] The tissue culture container 1 according to the second embodiment includes an outer container bottom 11 and an outer container peripheral wall portion 12 erected from a peripheral edge 11a of the outer container bottom 11, and an outer container 10 that encloses a first space 13 defined by the outer container bottom 11 and the outer container peripheral wall portion 12, a peripheral wall member 30 that is separably accommodated in the first space 13 and includes a plurality of connected well peripheral wall portions 31, and the outer container bottom 11 and the plurality of well peripheral wall portions 31 form a plurality of wells 40 each enclosing a plurality of second spaces 41, the second space 41 of each well 40 of the plurality of wells 40 is defined by one of a plurality of portions 11b of the outer container bottom 11 and one of the well peripheral wall portions 31 erected from a peripheral edge 11ba of one of the portions 11b, in each well 40, a gap 42 that communicates the second space 41 in each well 40 with the first space 13 is formed between at least a part in the circumferential direction of the peripheral edge 11ba of the portion 11b of the outer container bottom 11 and the well peripheral wall portion 31. It is characterized by this.

[0097] In the tissue culture container 1 according to the second embodiment, the culture solution stored in the first space 13 of the outer container 10 can flow into each second space 41 of the plurality of wells 40 through the gap 42 and fill the second space 41. Further, when the culture solution stored in the first space 13 of the outer container 10 is discharged by tilting the entire tissue culture container 1 or by sucking the culture solution, the culture solution in each second space 41 of the plurality of wells 40 can be simultaneously discharged through the gap 42. That is, according to the tissue culture container 1 according to the second embodiment, it is possible to exchange the culture solution in the plurality of wells 40 each containing the tissue T at one time in a short time. Further, it is possible to treat the tissue T contained in each of the plurality of wells 40 with the culture solution under uniform conditions.

[0098] Furthermore, when the peripheral wall member 30 is separated from the outer container 10 after the treatment with the culture solution using the tissue culture container 1 according to the second embodiment, the well 40 is disassembled and the plurality of tissues T are released into the first space 13 of the outer container 10, so that the plurality of tissues T can be collected at one time.

[0099] The gap 42 in the well 40 is formed between at least a part of the circumferential direction of the peripheral edge 11ba of the portion 11b of the outer container bottom 11 forming 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 through which the tissue T to be cultured cannot pass. For example, the width of the gap 42 is preferably 50% or less of the width of the tissue T to be cultured.

[0100] In the tissue culture container 1 according to the second embodiment, when observing the tissue T being cultured in the plurality of wells 40 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, the outer container bottom 11 of the outer container 10 is preferably made of a glass material having no autofluorescence.

[0101] Other features, usage methods, and modifications of the tissue culture container 1 according to the second embodiment are as described with respect to the tissue culture container 1 according to the first embodiment.

Claims

1. A tissue culture container for culturing tissues, comprising: an outer container having an outer container bottom and an outer container peripheral wall portion erected from the periphery of the outer container bottom, and enclosing a first space defined by the outer container bottom and the outer container peripheral wall portion; a bottom member detachably accommodated in the first space and having a plurality of connected well bottoms; a peripheral wall member detachably accommodated in the first space and having a plurality of connected well peripheral wall portions; wherein the plurality of well bottoms and the plurality of well peripheral wall portions form a plurality of wells, each enclosing a plurality of second spaces; the second space of each well of the plurality of wells is defined by one of the well bottoms and one of the well peripheral wall portions erected from the periphery of one of the well bottoms; in each well, a gap is formed between at least a part of the circumferential direction of the periphery of the well bottom and the well peripheral wall portion to communicate the second space in each well with the first space; a tissue culture container, characterized in that.

2. The tissue culture container according to claim 1, wherein the gap in each well is formed at the same circumferential position with respect to the well bottom of each well.

3. The tissue culture container according to claim 1 or 2, wherein 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 comprises a first engaging portion; the peripheral wall member further comprises a second engaging portion; the outer container comprises a third engaging portion that engages with the first engaging portion of the bottom member to position the bottom member on the outer container peripheral wall portion of the outer container, and a fourth engaging portion that engages with the second engaging portion of the peripheral wall member to position the peripheral wall member; at least one of the engagement between the first engaging portion of the bottom member and the third engaging portion of the outer container and the 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 be able to change the width of the gap in each well; the tissue culture container according to any one of claims 1 to 3.

5. At least one of the first engaging portion of the bottom member and the third engaging portion of the outer container is processable so as to be able to adjust the position of the bottom member with respect to the peripheral wall member, and / or At least one of the second engaging portion of the peripheral wall member and the fourth engaging portion of the outer container is machinable so that the position of the peripheral wall member with respect to the bottom member can be adjusted. The tissue culture container according to claim 4.

6. The first engaging portion of the bottom member and the third engaging portion of the outer container can engage at a plurality of positions so that the bottom member can be positioned at a plurality of different positions with respect 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 engage at a plurality of positions so that the peripheral wall member can be positioned at a plurality of different positions with respect to the bottom member. The tissue culture container according to claim 4.

7. Two or more of the bottom members are provided. Each of the two or more bottom members includes a first engaging portion that engages with the third engaging portion of the outer container to position the bottom member at a different position with respect to the peripheral wall member. The tissue culture container according to claim 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 position of the bottom member with respect to the peripheral wall member by adjusting the interval between the first engaging portion of the bottom member and the third engaging portion of the outer container, and / or A second spacer member that is removably interposed between the second engaging portion of the peripheral wall member and the fourth engaging portion of the outer container and adjusts the position of the peripheral wall member with respect to the bottom member by adjusting the interval between the second engaging portion of the peripheral wall member and the fourth engaging portion of the outer container is further provided The tissue culture container according to claim 4.

9. Two or more of the peripheral wall members are provided. Each of the two or more peripheral wall members includes a second engaging portion that engages with the fourth engaging portion of the outer container to position the peripheral wall member at a different position with respect to the bottom member. The tissue culture container according to claim 4.

10. The bottom member further includes a first engaging portion. The peripheral wall member further includes a second engaging portion that engages with the first engaging portion of the bottom member to position the peripheral wall member. The outer container includes a third engaging portion that engages with the first engaging portion of the bottom member on the outer container peripheral wall of the outer container to position the bottom member. At least one of the engagement between the first engaging portion of the bottom member and the third engaging portion of the outer container and the 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 be able to change the width of the gap in each well. The tissue culture container according to any one of claims 1 to 3.

11. Comprising N said peripheral wall members from the first peripheral wall member to the Nth peripheral wall member, The inner diameter of the (n - 1)th well peripheral wall portion, which is each of the plurality of well peripheral wall portions provided in the (n - 1)th peripheral wall member, is larger than the outer diameter of the nth well peripheral wall portion, which is each of the plurality of well peripheral wall portions provided in the nth peripheral wall member. The first peripheral wall member to the Nth peripheral wall member are removably combined so that the nth well peripheral wall portion of the nth peripheral wall member is inserted into the (n - 1)th well peripheral wall portion of the (n - 1)th peripheral wall member. N is an integer of 2 or more, and n is an integer of 2 or more and N or less. Each well is formed by one of the well bottoms and one of the combinations from the first well peripheral wall portion to the Nth well peripheral wall portion. The second space of each well is defined by one of the well bottoms and one of the combinations from the first well peripheral wall portion to the Nth well peripheral wall portion erected from the periphery of one of the well bottoms. In each well, the distance from the lower end of the (n - 1)th well peripheral wall portion to the well bottom is larger than the distance from the lower end of the nth well peripheral wall portion to the well bottom. The tissue culture container according to any one of claims 1 to 3.

12. Further comprising a lid that can cover the outer container, The bottom member further comprises a fifth engaging portion, The peripheral wall member further comprises a sixth engaging portion, The lid comprises a seventh engaging portion that engages with the fifth engaging portion of the bottom member to hold the bottom member, and an eighth engaging portion that engages with the sixth engaging portion of the peripheral wall member to hold the peripheral wall member. The tissue culture container according to any one of claims 1 to 11.

13. The tissue culture container 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.

14. A tissue culture container for culturing tissues, An outer container comprising an outer container bottom and an outer container peripheral wall portion erected from the periphery of the outer container bottom, and enclosing a first space defined by the outer container bottom and the outer container peripheral wall portion. A peripheral wall member including a plurality of connected well peripheral wall portions that are separably accommodated in the first space; comprising; the bottom of the outer container and the plurality of well peripheral wall portions form a plurality of wells, each enclosing a plurality of second spaces; the second space of each well 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 wall portions standing from the periphery of the one portion; in each well, 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, connecting the second space in each well and the first space; the width of the gap in each well is continuously and / or stepwise different depending on the circumferential position of the periphery of the portion of the bottom of the outer container in each well; A tissue culture container, characterized in that.

15. The tissue culture container according to claim 14, wherein the gap in each well is formed at the same circumferential position with respect to the portion of the bottom of the outer container in each well.

16. The peripheral wall member further includes a second engaging portion; the outer container includes a fourth engaging portion that engages with the second engaging portion of the peripheral wall member on the outer container peripheral wall portion of the outer container to position the peripheral wall member; at least one of the 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 be able to change the width of the gap in each well; The tissue culture container according to claim 14 or 15.

17. At least one of the second engaging portion of the peripheral wall member and the fourth engaging portion of the outer container is processable so that the position of the peripheral wall member with respect to the bottom of the outer container can be adjusted; The tissue culture container according to claim 16.

18. The second engaging portion of the peripheral wall member and the fourth engaging portion of the outer container can engage at a plurality of positions so that the peripheral wall member is positioned at a plurality of different positions with respect to the bottom of the outer container; The tissue culture container according to claim 16.

19. A second spacer member that is removably interposed between the second engaging portion of the peripheral wall member and the fourth engaging portion of the outer container, and adjusts the position of the peripheral wall member with respect to the bottom of the outer container by adjusting the interval between the second engaging portion of the peripheral wall member and the fourth engaging portion of the outer container; further comprising The tissue culture container according to claim 16.

20. comprising two or more of the peripheral wall members, each of the two or more peripheral wall members includes a second engaging portion that engages with the fourth engaging portion of the outer container to position the peripheral wall member at a different position with respect to the bottom of the outer container, The tissue culture container according to claim 16.

21. comprising N peripheral wall members from the first peripheral wall member to the Nth peripheral wall member, the inner diameter of the (n - 1)th well peripheral wall portion, which is each of the plurality of well peripheral wall portions included in the (n - 1)th peripheral wall member, is larger than the outer diameter of the nth well peripheral wall portion, which is each of the plurality of well peripheral wall portions included in the nth peripheral wall member, and the first peripheral wall member to the Nth peripheral wall member are removably combined such that the nth well peripheral wall portion of the nth peripheral wall member is inserted into the (n - 1)th well peripheral wall portion of the (n - 1)th peripheral wall member, N is an integer of 2 or more, and n is an integer of 2 or more and N or less, each well is formed by one of a plurality of portions of the bottom of the outer container and one of the combinations from the first well peripheral wall portion to the Nth well peripheral wall portion, the second space of each well is defined by one of a plurality of portions of the bottom of the outer container and one of the combinations from the first well peripheral wall portion to the Nth well peripheral wall portion erected from the periphery of the one portion, in each well, the distance from the lower end of the (n - 1)th well peripheral wall portion to the bottom of the outer container is larger than the distance from the lower end of the nth well peripheral wall portion to the bottom of the outer container, The tissue culture container according to claim 14 or 15.

22. further comprising a lid that can cover the outer container, the peripheral wall member further includes a sixth engaging portion, the lid includes an eighth engaging portion that engages with the sixth engaging portion of the peripheral wall member to hold the peripheral wall member, The tissue culture container according to any one of claims 14 to 21.

23. The tissue culture container according to any one of claims 14 to 22, wherein the bottom of the outer container of the outer container is made of a glass material.

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