Cell culture vessel, observation device, microscope, culture method, and observation method

Abstract

The cell culture vessel of the present invention is equipped with a plurality of three-dimensional compartments or a plurality of compartments surrounded by a frame corresponding to each side of a polyhedral shape, configured to be able to hold an aqueous liquid inside by surface tension. Adjacent compartments communicate with each other.

Description

Technical Field

[0001] The present invention relates to a cell culture container, an observation device, a microscope, a culture method, and an observation method.

Background Art

[0002] In recent years, attention has been focused on pharmaceutical development using organoids as a new in vitro experimental system. Organoids can reproduce complex biological systems, and the development of highly effective drugs is expected.

[0003] On the other hand, the three-dimensional culture technology of tissues such as organoids has not been established. In a general three-dimensional culture method, since only one type of extracellular matrix is used, it has been impossible to control the cell distribution of tissues. The extracellular matrix is diverse depending on the site even in one organ. Also, the differentiation, development, and localization of cells are determined by the position of the cells constituting the organ. Therefore, the three-dimensional arrangement of cells and cell body matrices is important, and various studies have been conducted so far.

[0004] For example, Non-Patent Document 1 describes the reproducibility of various in vivo phenomena by perfusing various tissues in a microfluidic device. Also, Non-Patent Document 2 describes a three-dimensional culture method using 3D bioprinting.

Prior Art Documents

Non-Patent Documents

[0005]

Non-Patent Document 1

Non-Patent Document 2

Summary of the Invention

[0006] However, the conventional technologies described above have the problem of requiring special equipment and techniques. Furthermore, setting the conditions is not easy. Therefore, there is a need for the development of a three-dimensional culture method that does not require special equipment and techniques and can be easily operated by anyone.

[0007] The present invention aims to realize a container for three-dimensional culture that does not require special equipment or techniques and can be easily operated by anyone. [Means for solving the problem]

[0008] The inventors, through diligent research to solve the above-mentioned problems, discovered that by changing the composition of the culture gel in which cells or cell tissues are embedded and the aqueous liquid placed around the culture gel, and by utilizing the differences in the surface tension of these aqueous liquids, the gel can be divided. In other words, despite being a simple procedure, the inventors found that it significantly improves the design flexibility of organoid culture, thus completing the present invention.

[0009] To solve the above problems, the present invention includes the following embodiments. A cell culture vessel comprising multiple three-dimensional compartments, each configured to retain aqueous liquid internally by surface tension, wherein adjacent compartments are interconnected. • A cell culture vessel comprising multiple compartments surrounded by a framework corresponding to each edge of a polyhedron, wherein the compartments are configured to hold aqueous liquid inside by surface tension, and adjacent compartments A method for culturing cells or cell tissue contained in a cell culture vessel having a polyhedral shape, wherein the cell culture vessel comprises a plurality of three-dimensional compartments configured to hold an aqueous liquid inside by surface tension, adjacent compartments communicating with each other, or a plurality of compartments surrounded by a skeleton corresponding to each edge of the polyhedron, the compartments configured to hold an aqueous liquid inside by surface tension, adjacent compartments communicating with each other, and the culture method comprises the step of containing a gel composition containing cells or cell tissue in the compartments. A method for observing cells or cell tissue contained in a cell culture vessel having a polyhedral shape, wherein the cell culture vessel comprises a plurality of three-dimensional compartments configured to hold an aqueous liquid inside by surface tension, adjacent compartments communicating with each other, or a plurality of compartments surrounded by a skeleton corresponding to each edge of the polyhedron, the compartments configured to hold an aqueous liquid inside by surface tension, adjacent compartments communicating with each other, and the cell culture vessel further comprises an axis portion extending outward from any of the vertices of the polyhedron shape in a direction not parallel to the normal of any of the faces of the polyhedron shape, and the observation method includes the step of rotating the cell culture vessel with the axis portion of the cell culture vessel as the axis of rotation. A method for observing cells or cell tissue contained in a cell culture vessel having a polyhedral shape, wherein the cell culture vessel comprises a plurality of three-dimensional compartments configured to hold an aqueous liquid inside by surface tension, adjacent compartments communicating with each other, or a plurality of compartments surrounded by a skeleton corresponding to each edge of the polyhedron, the compartments configured to hold an aqueous liquid inside by surface tension, adjacent compartments communicating with each other, and the fixing device for the cell culture vessel comprises a gripping part for gripping the cell culture vessel, and a shaft part connected to the gripping part, the shaft part extending outward from any of the vertices of the polyhedron shape in a direction not parallel to the normal of any of the faces of the polyhedron shape, and the observation method comprises the step of gripping the cell culture vessel with the fixing device and rotating the cell culture vessel with the shaft part as the axis of rotation. [Effects of the Invention]

[0010] According to one aspect of the present invention, it is possible to realize a container for three-dimensional cell culture or the like that does not require special devices and techniques and can be easily operated by anyone.

Brief Description of the Drawings

[0011] [Figure 1] It is a schematic diagram showing an example of the cell culture container of the present invention. [Figure 2] It is an enlarged view of a part of the cell culture container of FIG. 1. [Figure 3] It is an enlarged view of a part of the cell culture container of FIG. 1. [Figure 4] It is a view showing an example of the cell culture container of the present invention. [Figure 5] It is a view showing an example of the fixture of the observation device of the present invention. [Figure 6] It is a view showing a state where the fixture of FIG. 5 is attached to the cell culture container. [Figure 7] It is a perspective view showing a state where an example of the observation device of the present invention is installed on a microscope. [Figure 8] It is a view showing the result of Evaluation Example 1. [Figure 9] It is a view showing the result of Evaluation Example 2. [Figure 10] It is a view showing the result of Evaluation Example 3. [Figure 11] [[ID=4l]]It is a view showing the result on the 0th day of culture in Example 1. [Figure 12] It is a view showing the result on the 8th day of culture in Example 1. [Figure 13] It is a view showing the skeleton (frame) of a cell culture container whose cross section is substantially square. [Figure 14] It is a view showing the frame of a cell culture container whose cross section is substantially L-shaped. [Figure 15] It is a view showing the production of a cell culture container using the frame of a cell culture container whose cross section is substantially square. [Figure 16] It is a view showing the production of a cell culture container using the frame of a cell culture container whose cross section is substantially square. [Figure 17]It is a diagram showing the production of a cell culture container using a frame of a cell culture container with a substantially L-shaped cross section. [Figure 18] It is a diagram showing a frame of a cell culture container with a substantially L-shaped cross section. [Figure 19] It is a diagram showing the evaluation method of Evaluation Example 5. [Figure 20] It is a diagram showing the results of Evaluation Example 5. [Figure 21] It is a drawing related to the curvature of the interface between the hydrogel and air. [Figure 22] It is a drawing related to the curvature of the interface between the hydrogel and air. [Figure 23] It is a diagram showing the cell culture container produced in Evaluation Example 6. <​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​The cell culture vessel 10 is a container for containing and culturing cells or cell tissue. The cells or cell tissue are contained in the cell culture vessel 10 together with an aqueous liquid. In this specification, the aqueous liquid refers to the liquid before gelation. The aqueous liquid will be described later.

[0014] The cell culture container 10 according to the present invention has an external shape that is approximately cubic, as shown in Figure 1, for example. More specifically, the cell culture container 10 has an external shape that is approximately cubic, having 12 sides S1 to S12, as shown in Figure 1. Its height direction is called the z direction, the direction parallel to S4 or S12 is called the y direction, and the direction parallel to S1 or S9 is called the x direction. However, the shape of the cell culture container 10 is not limited to a cube, and may be a rectangular prism or other polyhedron or sphere. From the viewpoint of ease of handling of the cell culture container 10, a hexahedron is preferred as the shape (external shape) of the cell culture container 10, and a cube is more preferred. As for the size of the cell culture container 10, for example, the outer dimension of the outer diameter of the cell culture container 10 can be approximately 4 mm and the inner dimension can be approximately 3 mm, but the specific size of the cell culture container 10 is not limited to this embodiment. Note that the definitions of the x, y, and z directions are not limited to those stated above; they refer to three directions where the vectors representing each direction are mutually orthogonal, starting from the same origin.

[0015] (Container exoskeleton 11 and frame parts F1-F6) The cell culture vessel 10 is equipped with an exoskeleton 11 of the vessel corresponding to each side of its polyhedral (cubic) shape. The inside of the cell culture vessel 10 is divided into multiple compartments 100 and 200 by the exoskeleton 11 and frame sections F1 to F6. Frame sections F1 to F4 are frames equipped with a square outer frame (a U-shaped outer frame) having four sides and partitions that divide the inside of the outer frame into approximately nine equal parts in the vertical and horizontal directions. In other words, frame sections F1 to F4 are frames in which the inside of the outer frame is divided into nine approximately square mesh sections. Frame sections F5 and F6 are frames equipped with a U-shaped outer frame having three sides and partitions that divide the inside of the outer frame into approximately nine equal parts in the vertical and horizontal directions. In other words, frame sections F5 and F6 are U-shaped frames in which the inside of the outer frame is divided into nine approximately square mesh sections. The number, shape, and arrangement of the frame sections can be appropriately selected depending on how the inside of the cell culture vessel 10 is to be partitioned.

[0016] In terms of maintaining the surface tension of the aqueous liquid contained in the cell culture vessel 10, it is preferable to provide a frame so that each face of the polyhedral cell culture vessel 10 is divided into multiple sections, and it is even more preferable to provide a frame so that each face of the polyhedral shape is divided into multiple and equal sections. In the example of the cell culture vessel 10 shown in Figure 1, five faces are divided into nine approximately square mesh sections, and one face is divided into three approximately rectangular mesh sections.

[0017] The material for the exoskeleton 11 and frame parts F1 to F6 may be, for example, a biocompatible resin such as polycarbonate. An example of a resin is the resin used in 3D printers. The exoskeleton 11 and frame parts F1 to F6 are preferably treated with a surface treatment agent such as polydimethylsiloxane (PDMS) to increase the surface tension of the aqueous liquid contained in the cell culture vessel 10. Furthermore, the exoskeleton 11 and frame parts F1 to F6 are preferably transparent for ease of observation of cells or cell tissues.

[0018] (Sections 100 and 200) The cell culture vessel 10 is equipped with multiple compartments 100 and 200, each with a three-dimensional shape. Figure 2 shows three adjacent compartments 100 (compartments 101-103). Figure 3 shows one compartment 200. Each compartment 100 is surrounded by a skeleton SS1 corresponding to each side of a cube. Each compartment 200 is surrounded by skeletons SS10 and SS11 corresponding to each side of a rectangular prism. By providing auxiliary skeletons SS12 to the rectangular sides and bottom of compartment 200, each compartment 200, except for the top surface, is composed of surfaces partitioned in a roughly square mesh pattern. Note that each compartment 100-200 consists only of skeletons corresponding to each side of the three-dimensional shape, and the parts corresponding to the surfaces of the three-dimensional shape are open.

[0019] Each of the 100-200 compartments can hold aqueous liquid internally due to surface tension. This allows for the storage of aqueous liquid in units of 100-200 compartments. In other words, it is possible to store aqueous liquids of different compositions in each of the 100-200 compartments. From the perspective of holding aqueous liquid internally due to surface tension, the volume of one compartment is, for example, 0.001 to 10000 mm³. 3 Within the range of 1-500mm 3 Preferably within the range of 8 to 300 mm 3 It is more preferable that the lengths be within this range. In the case of a cubic compartment, such as compartment 100, the length of the framework SS1 corresponding to each side of the cube is, for example, in the range of 0.1 mm to 100 mm, preferably in the range of 1 mm to 10 mm, and more preferably in the range of 1 mm to 5 mm. In the case of a larger volume compartment, such as compartment 200, the amount of aqueous liquid that can be held by surface tension can be increased by constructing the sides and bottom surfaces with mesh-like partitions. When the sides and bottom surfaces are partitioned in a rectangular (approximately square, etc.) mesh pattern, the length of each side of the rectangle may be equal to the length of the framework SS1.

[0020] As shown in Figure 1, the compartments 100 are arranged in three directions: x, y, and z. The cell culture vessel 10 consists of a first layer with nine compartments 100 arranged without gaps in the x and y directions, and a second layer with nine compartments 100 arranged without gaps in the x and y directions. The cell culture vessel 10 further has a third layer on top of the second layer, with three compartments 200 arranged without gaps in the x and y directions. Adjacent compartments (for example, compartments 101 and 102) share their framework in adjacent regions and are in communication with each other. The number of compartments provided is illustrative and not particularly limited.

[0021] In terms of maintaining the surface tension of the aqueous liquid contained in the cell culture vessel 10 and ease of designing the three-dimensional arrangement, it is preferable that each compartment 100-200 be arranged in the x and y directions, the y and z directions, or the x and z directions, and more preferably arranged in the x, y and z directions.

[0022] The shape of each section 100-200 is not limited to a hexahedron (cube or rectangular prism), but may be any other polyhedron or spherical three-dimensional shape. From the viewpoint of maintaining the surface tension of the aqueous liquid, it is preferable that each section be cubic. The sizes of the sections may be the same or different. From the viewpoint of maintaining the surface tension of the aqueous liquid, it is preferable that each section be the same size.

[0023] As described above, each compartment 100-200 is configured to retain aqueous liquid inside due to surface tension. Furthermore, adjacent compartments (compartments 100 together, compartments 200 together, and compartments 100 and 200) are interconnected. This configuration allows for the cultivation of multiple different cells and / or extracellular matrix in an appropriate three-dimensional arrangement. This appropriate three-dimensional arrangement makes it possible to form biological systems such as angiogenesis systems and multi-organ interaction systems (e.g., the connection between the liver and bile ducts).

[0024] (Window section 12) The window portion 12 is surrounded by the 12 edges that constitute the exoskeleton 11. That is, the window portion 12 is composed of six faces that constitute the cubic shape shown in Figure 1 (i.e., faces composed of V1, V2, V3, V4; faces composed of V1, V2, V6, V5; faces composed of V2, V3, V7, V6; faces composed of V3, V4, V8, V7; faces composed of V1, V4, V8, V5; and faces composed of V5, V6, V7, V8). The material of the window portion 12 is preferably a light-transmitting material that allows nutrients, stimulating factors, etc. necessary for culturing cells or cell tissue (hereinafter also referred to as samples) contained in the cell culture vessel 10 to pass through. Specific materials for the window portion 12 preferably include, for example, agarose gel, polyacrylamide gel, sodium alginate, or collagen gel. In this way, by constructing the window portion 12 with a nutrient-permeable material, when the cell culture vessel 10 is immersed in a liquid culture medium, nutrients, stimulating factors, etc. contained in the liquid culture medium are supplied to the cells or cell tissue contained in the cell culture vessel 10 through the window portion 12. The window portion 21 may also play the role of a wall portion, which will be described later.

[0025] Furthermore, by constructing the window portion 12 with a light-transmitting material, the cells or cell tissue contained inside the cell culture vessel 10 can be observed from the directions of multiple faces that make up the cubic shape.

[0026] (Shaft portion 17) Furthermore, as shown in Figure 4, the cell culture vessel 10 may also include a shaft portion 17 that extends outward from any of the vertices of the polyhedron shape in a direction not parallel to the normal of any of the faces of the polyhedron shape.

[0027] As an example, as shown in Figure 1, the shaft portion 17 extends from one vertex V1 of the cubic cell culture vessel 10 in a direction along the straight line l connecting vertex V1 and vertex V7 located diagonally opposite to vertex V1. A bearing 18 may also be provided at vertex V7, which is located diagonally opposite to vertex V1.

[0028] Furthermore, the shaft portion 17 may, for example, extend along a straight line passing through the center of the cell culture container 10. Here, the center of the cell culture container 10 refers, for example, to the center of gravity of the cell culture container 10, but is not limited to this. For example, the center may refer to the intersection of multiple diagonals connecting the vertices of the polyhedral cell culture container 10.

[0029] Furthermore, the shape of the shaft portion 17 is not particularly limited, but as an example, it can be rod-shaped as shown in Figure 1. Also, the material of the shaft portion 17 may be a non-biocompatible material as long as it has a certain degree of strength.

[0030] The shaft portion 17 may be connected to a rotating mechanism, such as a stepping motor 38, which can rotate the cell culture container 10 in a predetermined direction and at a predetermined rotational speed using the shaft portion 17 as the axis of rotation. The rotational direction, rotational speed, and rotational duration of the stepping motor 38 are adjusted by the controller 39. In this embodiment, if the cell culture container 10 is rotated around the axis of the shaft portion 17 by a single-axis rotating mechanism, the cell culture container 10 can be observed with a microscope 40 while rotating around the axis of the shaft portion 17. However, in this embodiment, instead of the stepping motor 38, the observer may grasp the shaft portion 17 of the cell culture container 10 with tweezers or the like and rotate it manually.

[0031] (wall) Furthermore, the cell culture vessel 10 may have walls (not shown) on at least a portion of the faces of its polyhedral shape. The walls may cover the window 12, or the walls themselves may serve as the window 12. By providing walls, it is easier to maintain the shape of the gel composition contained in the cell culture vessel 10. In this specification, the gel composition refers to the aqueous liquid after gelation. In terms of injecting cells or cell tissue and / or aqueous liquid, it is preferable that at least one face of the polyhedral shape does not have walls.

[0032] Examples of materials that make up the wall include hydrogels such as agarose gel, polyacrylamide gel, sodium alginate, and collagen gel. The wall is preferably translucent for ease of observation of cells or cell tissue.

[0033] (aqueous liquid) Cells or cell tissues are housed in a cell culture vessel 10 together with an aqueous liquid. The aqueous liquid is a liquid in which water is the main solvent (the solvent in which water occupies the largest volume). The volume percentage of water in the solvent is preferably 50% or more, and may be more preferably 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%. The aqueous liquid can be any liquid capable of gelation. For example, the aqueous liquid may contain extracellular matrix such as collagen, laminin, entactin, proteoglycan, fibrin, fibronectin, and fibrinogen. The aqueous liquid may also contain TGF-β, fibroblast growth factor, tissue plasminogen activator, etc. The concentration of the extracellular matrix or each factor can be selected as appropriate. Matrigel (registered trademark) may be used as the aqueous liquid.

[0034] The cells may be a cell tissue having a structure in which they are aggregated in a certain pattern, or they may be cells that do not have such a tissue structure. Furthermore, cell tissue may grow by culturing cells that do not have a tissue structure.

[0035] The cell culture vessel 10 may contain two or more types of aqueous liquids. When containing two or more types of aqueous liquids, the cells or cell tissues contained in each aqueous liquid may be the same or different. The cell culture vessel may also contain an aqueous liquid that does not contain cells or cell tissues.

[0036] Even when two or more types of aqueous liquids are contained, each aqueous liquid is maintained within its respective compartment due to surface tension. After the aqueous liquids are placed in the cell culture vessel 10, gelation of the aqueous liquids occurs. This gelation prevents the gels from mixing between compartments. The compartmentalization of two or more gel compositions with different compositions can be achieved with the cell culture vessel of the present invention. By compartmentalizing two or more gel compositions with different compositions, three-dimensional culture of cells, cell tissues, or extracellular matrix can be initiated in an appropriate (intended) arrangement. Furthermore, by using the cell culture vessel 10, the operator can perform organoid culture with significantly improved design flexibility through the simple operation of injecting aqueous liquids. In addition, by compartmentalizing two or more gel compositions with different compositions, the morphology of cells, cell tissues, or extracellular matrix can be changed for each compartment.

[0037] To promote gelation, the cell culture vessel 10, which is filled with a gel composition containing cells or cell tissue, may be heated to a desired temperature in an incubator or the like. Other treatments to promote gelation include the addition of ions to the aqueous liquid and UV irradiation.

[0038] Cell culture may also be performed by placing a cell culture vessel 10, which is filled with a gel composition containing cells or cell tissue, into a liquid culture medium.

[0039] After culturing for a certain period, the cell culture vessel 10 covering the gel composition containing cells or cell tissue may be removed, and further culturing may be performed.

[0040] The cell culture vessel 10 can be manufactured, for example, by known technologies such as a 3D printer.

[0041] [Trimming of cell culture vessels] Modified examples of individual compartments constituting a cell culture vessel are shown in Figures 18 and 22, etc. These compartments are cubic in shape, and differ from the cubic compartments shown in Figures 1 and 2, etc., in that the cross-sections of each side of the cube (corresponding to the framework surrounding the compartment) are L-shaped (see also Figure 22). Note that the cross-section of each side of the cube refers to the cross-section obtained when each side is cut by a plane perpendicular to that side.

[0042] The compartment shown in Figure 18 is a cube shape composed of six square faces with one opening in the center. In Figure 18, W is the length of each side of the square opening (where the window section 12, described later, is provided). In Figure 18, S is the internal dimension of the compartment, which is the length of each side (external dimension) of the compartment minus 1 mm. In the compartment shown in Figure 18, the relationship S > W is satisfied.

[0043] From the standpoint of increasing surface tension by increasing the contact area between the aqueous liquid and the skeleton, and from the standpoint of observation such as imaging of cells in the cell culture vessel, W is preferably 1 mm to 4.0 mm, more preferably 1.5 mm to 3.5 mm, and even more preferably 2.0 mm to 3.0 mm. Also, S is preferably in the range of 1 mm to 10 mm, and more preferably in the range of 1 mm to 5 mm. Furthermore, the difference between S and W (SW) is preferably 0 to 6.0 mm, more preferably 0.5 mm to 5.5 mm, and even more preferably 1.0 mm to 5.0 mm.

[0044] It should be noted that an L-shaped cross-section is not limited to cases where the angle between the elements is approximately 90 degrees (see Figure 22); the angle between the elements can also be acute or obtuse (see Figure 27, etc.). In other words, the angle between the elements formed by an L-shaped cross-section varies depending on the shape of the section.

[0045] Each of the six square faces that make up the section shown in Figure 18 can also be considered as a square face having an opening surrounded by an edge of a predetermined width. In the case of Figure 18, the predetermined width of the edge is (SW) × 1 / 2 + 0.5 mm, where S and W are dimensions in millimeters.

[0046] The individual compartments shown in Figures 18 and 22, etc., can be arranged in multiples, similar to the compartments shown in Figures 1 and 2, etc., to constitute a cell culture vessel. Alternatively, if necessary, a single cell culture vessel may be constructed by combining the types of compartments shown in Figures 18 and 22, etc., with the compartments shown in Figures 1 and 2, etc.

[0047] [2. Observation equipment and microscope] An example of an observation apparatus and microscope according to the present invention will be described in detail with reference to Figures 5-7.

[0048] As shown in Figure 7, the observation device 30 comprises a cell culture vessel 10 with a shaft portion 17 and a rotation mechanism (stepping motor 38). By installing the observation device 30 on the microscope 40, time-lapse images of cells or cell tissue can be acquired.

[0049] Figure 7 is a perspective view showing the observation device 30 according to Embodiment 1 of the present invention installed on a microscope 40. The coordinate axes shown in Figure 7 represent a stationary coordinate system expressed as XYZ axes with the Z axis in the vertical direction. The observation device 30 according to Embodiment 1 of the present invention and the microscope 40 on which the observation device 30 is installed will be described below with reference to Figure 7.

[0050] The observation device 30 comprises a cell culture vessel 10 and a rotation mechanism that grips the shaft portion 17 of the cell culture vessel 10 and rotates the cell culture vessel 10 using the shaft portion 17 as the axis of rotation.

[0051] More specifically, as shown in Figure 7, the observation apparatus 30 according to this embodiment comprises a Z-axis fine-adjustment stage 31, an oblique fixing holder 34, a rotating stage 35, an extension unit 36, a collet chuck 37, a stepping motor 38, and a cell culture vessel 10. Here, the rotation mechanism described above is configured, for example, to include an oblique fixing holder 34, an extension unit 36, a collet chuck 37, and a stepping motor 38.

[0052] The observation device 30 according to this embodiment is placed on the microscope stage 42 of the microscope 40.

[0053] The Z-axis fine-adjustment stage 31 of the observation device 30 comprises a base 32 placed on the microscope stage 42 of the microscope 40, and a pedestal portion 33 extending vertically upward from the base 32. The height of the pedestal portion 33 in the Z-axis direction can be adjusted. This allows for fine adjustment of the Z-axis position of the cell culture container 10, which is held by the collet chuck 37 described later.

[0054] The oblique fixing holder 34 is a plate-shaped member. The oblique fixing holder 34 is connected at an angle to the base portion 33 of the Z-axis fine adjustment stage 31. The angle between the oblique fixing holder 34 and the base portion 33 of the Z-axis fine adjustment stage 31 is set so that the normal to the surface formed by at least one of the windows of the cell culture container 10 is parallel to the optical axis of the lens barrel 41. As an example, when using a cell culture container 10 having a cubic shape, as shown in Figure 7, the oblique fixing holder 34 is connected to the upper side of the base portion 33 of the Z-axis fine adjustment stage 31 at an angle of 135 degrees to the base portion 33 of the Z-axis fine adjustment stage 31. That is, in the embodiment shown in Figure 7, the oblique fixing holder 34 is fixed to the base portion 33 of the Z-axis fine adjustment stage 31 at an angle of 45 degrees with respect to the horizontal direction (XY direction).

[0055] A disc-shaped rotating stage 35 is provided on the lower surface of the oblique fixing holder 34. The rotating stage 35 is driven by a stepping motor 38 to rotate in both clockwise and counterclockwise directions. As the rotating stage 35 rotates, the member attached to the tip of the rotating stage 35 can be rotated in either clockwise or counterclockwise directions. The rotating stage 35 may be configured to rotate automatically using the stepping motor 38 as described above, but it may also be configured to be rotated manually by an observer.

[0056] An extension portion 36 extends linearly from the center of the rotating stage 35 in a direction perpendicular to the oblique fixing holder 34 and the rotating stage 35, i.e., at a 45-degree angle to the horizontal direction (XY direction). A collet chuck 37 is connected to the tip of the extension portion 36. The collet chuck 37 grips the shaft portion 17 of the cell culture vessel 10. For example, the collet chuck 37 with the cell culture vessel 10 attached may be held at a 45-degree angle to the horizontal plane, and the cell culture vessel 10 may be held in the horizontal direction. As shown in Figure 7, the cell culture vessel 10 is attached to the collet chuck 37 while immersed in the liquid culture medium of the dish (or well) 51.

[0057] Alternatively, instead of attaching the cell culture container 10 to the collet chuck 37, a fixing device 20 to which a cell culture container 10 without a shaft portion 17 is attached may be attached to the extension portion 36.

[0058] Therefore, as the rotating stage 35 rotates, the cell culture container 10 attached to the tip of the collet chuck 37 can be rotated at a predetermined speed with the shaft portion 17 as the axis of rotation, while maintaining the extension portion 36 at a 45-degree angle with respect to the horizontal direction. The rotation speed and rotation duration of the rotating stage 35 (i.e., the rotation speed, stop time, and rotation duration of the cell culture container 10) can be set arbitrarily, and continuous rotation and intermittent operation with repeated rotation and stopping are also possible. For example, the cell culture container 10 may be set to rotate clockwise at a speed of 2 times / minute (i.e., 120 degrees of rotation in 10 seconds), and the rotation duration may be set to 3 days while repeating the cycle of rotating 120 degrees, stopping for 110 seconds, and then rotating 120 degrees clockwise again.

[0059] <Observation of cells or cell tissue using a microscope> The following describes a method of observing a cell culture vessel 10 by immersing it in a liquid culture medium and connecting it to an observation device 30, then placing it on a microscope 40. The microscope 40 can be a conventionally used microscope. For example, a confocal laser microscope or a two-photon microscope can be used as the microscope 40. Also, while Figure 7 uses an inverted microscope as an example, an upright microscope may also be used.

[0060] As shown in Figure 7, the cell culture vessel 10 is immersed in the liquid culture medium in the dish (or well) 51.

[0061] When observing the cell culture vessel 10, the cell culture vessel 10 is immersed in the dish 51 and placed on the microscope stage 42 so that the cells or cell tissue contained in the cell culture vessel 10 are within the field of view of the microscope tube 41 of the microscope 40.

[0062] Next, the Z-axis fine adjustment stage 31 of the observation device 30 is placed in a predetermined position on the microscope stage 42 of the microscope 40. Then, the tip of the extension part 36 of the observation device 30 is connected to the cell culture container 10, which is horizontally immersed in the dish 51, via the collet chuck 37. After that, the Z-axis fine adjustment stage 31 is used to fine-tune the position of the cell culture container 10 in the Z-axis direction.

[0063] Once the cell culture vessel 10 is positioned, the rotation speed of the rotating stage 35, the stop time for intermittent operation, and the rotation duration are set, and the process of observing cells or cell tissue (i.e., acquiring images) while the rotation is stopped is repeated.

[0064] The above description concerns a cell culture vessel 10 equipped with a shaft portion 17. However, a cell culture vessel 10 without a shaft portion 17 may also be used for observing cells or cell tissues.

[0065] The following describes the fixing device 20 used to fix a cell culture vessel 10 that does not have a shaft portion 17. The cell culture vessel 10 has the same configuration as the cell culture vessel 10 described above, except that it does not have a shaft portion 17. Figure 5 shows an example of the fixing device 20 used to fix a cell culture vessel 10 that does not have a shaft portion 17, and Figure 6 shows an example of the fixing device 20 attached to the cell culture vessel 10.

[0066] (Fixing fixture) As shown in Figure 5, the fixing device 20 includes a gripping portion 22 for gripping the cell culture container 10 and a shaft portion 25 connected to the gripping portion 22.

[0067] The gripping portion 22 further comprises three gripping portions 22a, 22b, and 22c that extend in directions perpendicular to each other and have substantially equal lengths. The tips of the three gripping portions 22a, 22b, and 22c are each provided with claws 22d, 22e, and 22f.

[0068] As shown in Figure 6, when the fixing device 20 is attached to the cell culture vessel 10, the gripping parts 22a, 22b, and 22c are positioned along the parts of the exoskeleton 11 that correspond to the three sides (the sides connecting V1 and V4, V1 and V2, and V1 and V5) that form the vertex V1 of the cell culture vessel 10. In addition, the claws 22d, 22e, and 22f grip the vertex V1 of the cell culture vessel 10 and the adjacent vertices V4, V2, and V5 of the exoskeleton, respectively.

[0069] When the fixing device 20 is fixed to the cell culture vessel 10, the shaft portion 25 of the fixing device 20 extends outward from the vertex V1 of the polyhedron shape (cube shape) of the cell culture vessel 10 in a direction that is not parallel to the normal of any face of the polyhedron shape.

[0070] When observing cells or cell tissue contained in the cell culture vessel 10, the cell culture vessel 10 is rotated using the shaft portion 25 of the fixing device 20 as the axis of rotation. This allows for the acquisition of high-resolution images of cells or cell tissue in the Z-axis direction, similar to the case of a cell culture vessel 10 equipped with a shaft portion 17.

[0071] [3.Culture method] The culture method of the present invention is a method for culturing cells or cell tissue contained in the cell culture vessel described above. The culture method of the present invention includes a step of containing a gel composition containing cells or cell tissue in a compartment 100 of the cell culture vessel 10 (hereinafter sometimes abbreviated as the containing step).

[0072] (Storage process) In the containment step, for example, an aqueous liquid containing cells or cell tissue is contained in a compartment 100 of the cell culture vessel 10, and as the aqueous liquid gels, a gel composition containing cells or cell tissue is contained in the compartment 100. Alternatively, an aqueous liquid that does not contain cells or cell tissue may be contained in the compartment 100 of the cell culture vessel 10.

[0073] (Other processes) The culture method of the present invention may include steps other than the containment step. Examples of other steps include a wall formation step.

[0074] (Wall forming process) In the wall formation step, walls are formed on at least a portion of the faces of the polyhedral shape of the cell culture container 10. For example, as shown in the example, walls can be formed by placing the cell culture container 10 in liquid wall material heated to a certain temperature and gelling the liquid wall material. It is preferable to perform the wall formation step before the containment step.

[0075] [4. Observation Method] The observation method of the present invention is a method for observing cells or cell tissue contained in the cell culture vessel described above. The observation method of the present invention includes a step of rotating the cell culture vessel 10 with respect to the shaft portion 17 if the cell culture vessel 10 is provided with a shaft portion 17 (hereinafter sometimes abbreviated as the first rotation step). If the cell culture vessel 10 is not provided with a shaft portion, the method includes a step of gripping the cell culture vessel 10 with a fixing device 20 and rotating the cell culture vessel 10 with respect to the shaft portion of the fixing device (hereinafter sometimes abbreviated as the second rotation step).

[0076] [5. Summary of Embodiments] Embodiments of the present invention include, for example, the following aspects. <1> A cell culture vessel comprising multiple three-dimensional compartments, each configured to retain aqueous liquid internally by surface tension, wherein adjacent compartments are interconnected. <2> A cell culture vessel comprising a plurality of compartments surrounded by a framework corresponding to each edge of a polyhedron, wherein the compartments are configured to hold aqueous liquid inside by surface tension, and adjacent compartments are in communication with each other. <3> The cell culture vessel has a polyhedral shape, and further comprises translucent windows provided at positions corresponding to each of the multiple faces of the polyhedral shape. <1> or <2> The cell culture vessel described above. <4> The cell culture vessel has a polyhedral shape, and further comprises an axis extending outward from any of the vertices of the polyhedral shape in a direction not parallel to the normal of any of the faces of the polyhedral shape. <1> ~ <3> A cell culture vessel as described in any one of the following. <5> The above sections are arranged side by side in at least two directions selected from the X, Y, and Z directions. <1> ~ <4> A cell culture vessel as described in any one of the following. <6> The above section is surrounded by a framework corresponding to each edge of a hexahedron. <1> ~ <5> A cell culture vessel as described in any one of the following. <7> The cross-section of the skeleton corresponding to each edge of the above polyhedron is L-shaped. <1> ~ <6> A cell culture vessel as described in any one of the following items. <8> At least a portion of the above compartment is filled with a gel composition containing cells or cell tissue. <1> ~ <7> A cell culture vessel as described in any one of the following items. <9> Filled with two or more of the above gel compositions, <8> The cell culture vessel described above. <10> Its external shape is polyhedral. <1> ~ <9> An observation device comprising a cell culture vessel as described in any one of the above, a fixing device for the cell culture vessel, and a rotation mechanism for rotating the cell culture vessel, wherein the fixing device comprises a gripping portion for gripping the cell culture vessel, and a shaft portion connected to the gripping portion, the shaft portion extending outward from any of the vertices of the polyhedron shape in a direction not parallel to the normal of any of the faces of the polyhedron shape, and the rotation mechanism grips the shaft portion and rotates the cell culture vessel gripped by the fixing device with the shaft portion as the axis of rotation. <11> Its external shape is polyhedral. <4> An observation device comprising a cell culture vessel as described above, and the shaft portion of the cell culture vessel, which is grasped and rotated using the shaft portion as the axis of rotation. <12> <10> or <11> A microscope equipped with the observation device described above. <13> A method for culturing cells or cell tissue contained in a cell culture vessel having a polyhedral shape, wherein the cell culture vessel comprises a plurality of three-dimensional compartments configured to hold an aqueous liquid inside by surface tension, adjacent compartments communicating with each other, or a plurality of compartments surrounded by a skeleton corresponding to each edge of the polyhedron, the compartments configured to hold an aqueous liquid inside by surface tension, adjacent compartments communicating with each other, and the culture method comprises the step of containing a gel composition containing cells or cell tissue in the compartments. <14> A method for observing cells or cell tissue contained in a cell culture vessel having a polyhedral shape, wherein the cell culture vessel comprises a plurality of three-dimensional compartments configured to hold an aqueous liquid inside by surface tension, adjacent compartments communicating with each other, or a plurality of compartments surrounded by a skeleton corresponding to each edge of the polyhedron, the compartments configured to hold an aqueous liquid inside by surface tension, adjacent compartments communicating with each other, and the cell culture vessel further comprises an axis portion extending outward from any of the vertices of the polyhedron shape in a direction not parallel to the normal of any of the faces of the polyhedron shape, and the observation method includes the step of rotating the cell culture vessel with the axis portion of the cell culture vessel as the axis of rotation. <15> A method for observing cells or cell tissue contained in a cell culture vessel having a polyhedral shape, wherein the cell culture vessel comprises a plurality of three-dimensional compartments configured to hold an aqueous liquid inside by surface tension, with adjacent compartments communicating with each other, or comprising a plurality of compartments surrounded by a skeleton corresponding to each edge of the polyhedron, the compartments configured to hold an aqueous liquid inside by surface tension, with adjacent compartments communicating with each other, and the fixing device for the cell culture vessel comprises a gripping part for gripping the cell culture vessel, and a shaft part connected to the gripping part, the shaft part extending outward from any of the vertices of the polyhedron shape in a direction not parallel to the normal of any of the faces of the polyhedron shape, and the observation method comprises the step of gripping the cell culture vessel with the fixing device and rotating the cell culture vessel with the shaft part as the axis of rotation. [Examples]

[0077] In the following examples, unless otherwise specified, % represents mass %.

[0078] [Evaluation Example 1] Evaluation by preparing cell culture vessels and filling them with three types of gel compositions. A cell culture vessel 10 (1 cm on each side) was fabricated as shown in Figure 1. The cell culture vessel 10 was fabricated using a 3D printer (manufactured by Keyence Corporation). As shown in Figure 1, the inside of the cell culture vessel 10 is divided into 27 equal sections. Each of the divided sections is roughly cubic in shape and is interconnected as shown in Figure 1.

[0079] Nine compartments, including compartment 101, were filled with Matrigel® mixed with blue food coloring using a pipette. Nine compartments, including compartment 102, were filled with Matrigel® mixed with fluorescent beads using a pipette. Nine compartments, including compartment 103, were filled with Matrigel® using a pipette.

[0080] After filling all compartments, the gel-filled frames were placed in an incubator at 37°C for approximately 25 minutes, before the gels could adhere to each other and begin to mix through diffusion.

[0081] Figure 8 shows the cell culture vessels after incubator storage. It was observed that the three different gel compositions were separated into distinct sections.

[0082] [Evaluation Example 2] Evaluation by preparing cell culture vessels and filling them with two types of gel compositions. As shown in Figure 9, a rectangular cell culture vessel 70 (4 mm long x 8 mm wide x 4 mm high) with two compartments of the same size was fabricated. The frame was made using a 3D printer. Each compartment is roughly cubic in shape and interconnected.

[0083] One compartment 71 (left side of Figure 9) was filled with Matrigel® mixed with blue food coloring using a pipette. The other compartment 72 (right side of Figure 9) was filled with Matrigel® mixed with yellow food coloring using a pipette.

[0084] After filling all compartments with the gel composition, the gel-filled frames were placed in an incubator and stored at 37°C for approximately 25 minutes, before the gels could adhere to each other and begin to mix through diffusion.

[0085] Figure 9 shows the cell culture vessels after incubator storage. It was observed that the two types of gel compositions were separated.

[0086] [Evaluation Example 3] Evaluation by preparing cell culture vessels and filling them with two types of gel compositions. As shown in Figure 10, a cubic cell culture vessel (1 cm long x 1 cm wide x 0.5 cm high) with four compartments was fabricated. The frame was made using a 3D printer. Each compartment is interconnected. The frames used in evaluation examples 4-6 below were also all made using a 3D printer.

[0087] In Figure 10, compartment 81 was filled with Matrigel® mixed with blue food coloring using a pipette. Compartment 82 was filled with Matrigel® mixed with red food coloring using a pipette. Compartment 83 was filled with Matrigel® mixed with red food coloring using a pipette. Compartment 84 was filled with Matrigel® mixed with red food coloring using a pipette.

[0088] After filling all compartments with the gel composition, the gel-filled frames were placed in an incubator and stored at 37°C for approximately 25 minutes, before the gels could adhere to each other and begin to mix through diffusion.

[0089] Figure 10 shows the cell culture vessels after incubator storage. It was observed that the two types of gel compositions were separated.

[0090] [Example 1] Cell culture using a cell culture vessel (1) Construction of the wall section (exterior wall) Approximately 20-200 μL of liquid 1.5% agarose, warmed to 85°C, was dropped onto a glass slide. Next, the cell culture vessel prepared in Evaluation Example 1 was placed on top of the agarose, and the glass slide was cooled to gel the agarose.

[0091] After the agarose gelled, the excess agarose that protruded from the frame was cut and removed, creating an agarose outer wall on the frame. Of the six sides of the frame, an agarose outer wall was created on five sides.

[0092] (2) Filling with HUVEC-containing gel Sections 101 and 103 in Figure 1 were filled with 50 μL of HUVEC (Human Umbilical Vein Endothelial Cell)-containing gel. Matrigel®, containing 40% collagen, was used as the gel. The HUVEC-containing gel was filled using a pipette from the frame side where the agarose outer wall had not been prepared.

[0093] (3) Filling with HepG2-HFL-containing gel 50 μL of HepG2-HFL spheroid-containing gel was packed into compartment 102 in Figure 1. HepG2-HFL spheroids are spheroids composed of mHepG2 (human liver cancer-derived cells) and HFL (human fibroblasts). Matrigel®, containing 42.4% collagen and 15.2% fibrin, was used as the gel. The HepG2-HFL spheroid-containing gel was packed using a pipette from the side of the frame where the agarose outer wall had not been prepared.

[0094] (4) Gel crosslinking and cell culture After filling the internal space of the frame with gel, the gel-filled frame was placed in an incubator at 37°C for approximately 25 minutes before the gels could adhere to each other and begin to mix through diffusion. After confirming that the gels had cross-linked, the frame was placed in a liquid culture medium and cells were cultured in a 37°C incubator for 8 days.

[0095] Figure 11 shows a magnified image of the frame after 0 days of culture, and Figure 12 shows a magnified image of the frame after 8 days of culture. As shown in Figures 11 and 12, it was found that by using the cell culture vessel 10 prepared in Evaluation Example 1 and changing the composition of the aqueous liquid in the center and on both sides of the frame, heterogeneous extracellular matrices can be separated by the difference in surface tension of the aqueous liquid. It was also found that heterogeneous cells can be separated by changing the cells or cell tissues cultured in the center and on both sides of the frame.

[0096] Furthermore, although the extracellular matrix was incorporated into the gel in this embodiment, it is conceivable that the initial position or shape of the cells could be controlled by packing the cells instead of the extracellular matrix.

[0097] [Evaluation Example 4] Examination of Frame Shape Figure 13 shows a cell culture vessel frame (5 mm per side) with a roughly rectangular cross-section in the vertical direction. Hereafter, the frame in Figure 13 may be referred to as a straight frame. Figure 14 shows a cell culture vessel frame (5 mm per side) with a roughly L-shaped cross-section in the vertical direction. Hereafter, the frame in Figure 14 may be referred to as an L-shaped frame.

[0098] In Figure 13, a frame containing 30% collagen (hereinafter referred to as "collagen gel 1") was filled into the frame without creating an agarose outer wall, and the incubator was stored at 37°C for approximately 25 minutes. After incubator storage, when the prepared cell culture vessel was lifted while still filled in the frame, collagen gel 1 sometimes detached from the frame, depending on the viscosity of the agarose outer wall, as shown in the right side of Figure 13. On the other hand, when the prepared cell culture vessel was lifted after filling the frame with collagen gel 1 without creating an agarose outer wall in Figure 14 and storing it in an incubator at 37°C for approximately 25 minutes, collagen gel 1 did not detach from the frame, as shown in the right side of Figure 14. This suggests that the contact area between the gel and the frame increased and the surface tension increased by making the frame L-shaped.

[0099] Figures 15 and 17 show the results of filling the frame of a cell culture vessel without an agarose outer wall with collagen gel 1. Figure 16 shows the results of filling the frame of a cell culture vessel with an agarose outer wall with collagen gel 1. Figures 15 and 16 used a straight frame with nine roughly cubic compartments of the same size (each compartment having a side length of 5 mm). Figure 17 used an L-shaped frame with nine roughly cubic compartments of the same size (each compartment having a side length of 5 mm). In Figures 15-17, three types of collagen gel 1 (collagen gel 1 mixed with red, blue, and yellow food coloring, respectively) were filled into each compartment.

[0100] As shown in Figures 15 and 16, in the case of a straight frame, the collagen gel 1 could not be maintained within the compartment unless an agarose outer wall was fabricated. Also, as shown in Figure 16, although the collagen gel 1 could be maintained within the compartment, after approximately 25 minutes of storage in a 37°C incubator, the three colors of collagen gel 1 mixed together, and it was not possible to localize each color of collagen gel 1 into the desired compartment. This is thought to be due to the significant influence of gravity. On the other hand, as shown in Figure 17, when an L-shaped frame was used, it was possible to localize each color of collagen gel 1 into the desired compartment.

[0101] [Evaluation Example 5] Conditional Study of L-shaped Frame Next, we evaluated the physical forces that counteract the effects of gravity on the L-shaped frame. We fabricated an L-shaped frame with a roughly cubic shape as shown in Figure 18. The frame width was fixed at 1 mm per side, and frames were fabricated with W ranging from 2.0 mm to 4.0 mm and S ranging from 3.0 mm to 7.0 mm in Figure 18.

[0102] Next, collagen gel 1 was filled into the L-shaped frame shown in Figure 18, and as shown in Figure 19, the prepared cell culture vessel was lifted while still filled in the frame, and the size of the droplet formed at the bottom of the cell culture vessel (height of the droplet from the bottom of the cell culture vessel) was measured. The measurement results are shown in Figure 20. Frames with W=4.0mm and S=4.0mm were subjected to two experiments (n=2), and all other frames were subjected to three experiments (n=3).

[0103] In Figure 20, the vertical axis represents the length of W in Figure 18, and the horizontal axis represents the length of S in Figure 18. In Figure 20, "○" indicates that no droplets were observed. "##" indicates that droplets were observed. "×" indicates that droplets dripped, and the height of the droplets could not be measured.

[0104] As shown in Figure 20, it was found that droplet formation is more likely to occur (collagen gel 1 is more likely to detach from the frame) when both W and S exceed a certain range.

[0105] Figures 21 and 22 show the results of evaluating the curvature of the interface between the gel composition and air. Furthermore, it was found that the initial shape of the gel composition filled into the L-shaped frame (initial hydrogel shape from pipette) and the shape of the gel composition at equilibrium (hydrogel shape at equilibrium) should be considered when designing the L-shaped frame. These evaluations suggest that in the L-shaped frame, the effect of weight is hindered by the interaction of physical forces, and that Laplace pressure is important.

[0106] [Evaluation Example 6] Evaluation by preparing cell culture vessels and filling them with three types of gel compositions. Using an L-shaped frame, cell culture vessels of various shapes were fabricated. Three types of collagen gel 1 (collagen gel 1 mixed with red, blue, and yellow food coloring, respectively) were filled into each compartment of the frame, and the vessels were incubated at 30°C for approximately 25 minutes. The cell culture vessels after incubation are shown in Figures 23-28. As shown in Figure 1, each compartment of the cell culture vessel is interconnected.

[0107] Figure 23 shows a cell culture vessel made from a frame with five compartments, measuring S=4.0mm and W=3.0mm. Figure 24 shows a cell culture vessel made from a frame with nine compartments, measuring S=4.0mm and W=2.5mm. Each compartment of the frames in Figures 23 and 24 is approximately cubic in shape.

[0108] Figure 25 shows a cell culture vessel made from a frame with 27 compartments, each measuring S=3.0 mm and W=2.0 mm. The cell culture vessel in Figure 25 is further tilted at a desired angle along the diagonal axis by a roughly cubic fixing device. Each compartment of the frame in Figure 25 is roughly cubic in shape. As shown in Figure 25, tilting the cell culture vessel along the diagonal axis so that gravity pulls it towards the edge or corner prevented the formation of droplets of the desired aqueous liquid and prevented easy mixing with adjacent aqueous liquids.

[0109] Figure 26 shows a cell culture vessel made from a frame that is roughly square-pyramidal in shape, with roughly triangular pyramidal compartments with sides of 5 mm. Figure 27 shows a cell culture vessel made from a rectangular frame (5 mm long x 3 mm wide x 2.5 mm high) with eight compartments of the same size. Figure 28 shows a cell culture vessel made from a hexagonal prism frame (base side length 3 mm, height 6 mm) with twelve roughly triangular prism compartments of the same size.

[0110] [Example 2] Formation of bronchi using a cell culture vessel A cell culture vessel was prepared using three L-shaped frames with dimensions S=4.0 mm and W=3.0 mm. Of the three compartments in Figure 29, the leftmost compartment (compartment 1) was filled with Matrigel® only. The middle compartment (compartment 2) was filled with Matrigel® containing 0.15 mM genipine (crosslinking agent). The rightmost compartment (compartment 3) was filled with Matrigel® containing 0.3 mM genipine. In filling compartment 3, normal human bronchial epithelial cells (NHBE) or mouse endothelial cells were also used.

[0111] After filling, cells were injected into the gels before the gels adhered to each other and before the different gels began to mix by diffusion. The gel-filled frames were then placed in an incubator and stored at 37°C for approximately 25 minutes. After confirming that the gels had cross-linked, the frames were placed in liquid culture medium and cultured for 96 hours (human lung cells) or 92 hours (mouse endothelial cells).

[0112] Figure 30 shows the culture results of human bronchial epithelial cells, and Figure 31 shows the culture results of mouse endothelial cells. As can be seen from Figures 30 and 31, bronchi formed by culture were observed in both the left and middle compartments. The left compartment, where genipin was not added, showed the most bronchial branching, and the number of bronchial branches decreased as the concentration of genipin, a crosslinking agent, increased. Also, the length of the bronchial branches was longest in the left compartment where genipin was not added, and the length of the bronchial branches decreased as the concentration of genipin increased. From these results, it was found that the morphology of tissue can be altered in tissue culture by changing the viscosity of the aqueous liquid filled in each compartment of the cell culture apparatus.

[0113] From the above, it was found that by using the cell culture vessel (frame) prepared in this embodiment, it is possible to start three-dimensional culture with appropriate cells and cell matrix in an appropriate arrangement, and the design freedom of organoid culture can be greatly improved. Furthermore, it was found that by using the frame prepared in this embodiment, three-dimensional culture can be performed by the simple operation of filling the frame with a gel containing cells or cell tissue, and no special equipment or techniques are required. [Industrial applicability]

[0114] The present invention provides a screening system for highly effective therapeutic drugs, which can be used, for example, in pharmaceutical applications. [Explanation of symbols]

[0115] 10, 70, 80 cell culture vessels 11 Exoskeleton 12 Window section 15 Convex part 16 Tip 17. Axis (of the cell culture vessel) 18 bearings 20 Fixtures 22 Gripping part 25 (Shaft of the fastener) 30 Observation device 31 Z-axis fine adjustment stage 32 bases 33 Base 34. Oblique fixing holder 35-rotation stage 36 Stretching section 37 Collet Chuck 38 Stepping motor 39 Controllers 41 Telescope Tube 42 Microscope Stages 51 Dishes 60 Light Sheet Microscopes 61 Miller 62 Light Seat 63 Laser illumination section Plots 71, 72, 81-84, 100-104, and 200

Claims

1. A cell culture vessel, It has multiple compartments surrounded by a framework corresponding to each edge of a polyhedron, The above compartment is configured to be able to hold aqueous liquid inside by surface tension. Adjacent plots are connected to each other, The volume of the above compartment is 1 to 500 mm³. Cell culture vessel.

2. The cell culture container according to claim 1, wherein the outer shape of the cell culture container is polyhedral, and further comprises translucent windows provided at positions corresponding to each of the multiple faces of the polyhedral shape.

3. The cell culture container according to claim 1 or 2, wherein the outer shape of the cell culture container is polyhedral, and further comprises an axis portion extending outward from any of the vertices of the polyhedral shape in a direction not parallel to the normal of any of the faces of the polyhedral shape.

4. The cell culture vessel according to any one of claims 1 to 3, wherein the above-mentioned compartments are arranged side by side in at least two directions selected from the X, Y, and Z directions.

5. The cell culture vessel according to any one of claims 1 to 4, wherein the above compartment is surrounded by a skeleton corresponding to each edge of a hexahedron.

6. A cell culture vessel according to any one of claims 1 to 5, wherein the cross-section of the skeleton corresponding to each edge of the polyhedron is L-shaped.

7. A cell culture vessel according to any one of claims 1 to 6, wherein at least a portion of the above compartment is filled with a gel composition containing cells or cell tissue.

8. A cell culture vessel according to claim 7, which is filled with two or more of the above-mentioned gel compositions.

9. An observation device comprising a cell culture vessel according to any one of claims 1 to 8, having a polyhedral shape externally, a fixing device for the cell culture vessel, and a rotating mechanism for rotating the cell culture vessel, The above-mentioned fastener is, A gripping part for gripping the cell culture vessel, The device comprises a shaft portion connected to the gripping portion, which extends outward from any of the vertices of the polyhedron shape in a direction not parallel to the normal of any of the faces of the polyhedron shape, The above-described rotation mechanism is an observation device that grips the shaft portion and rotates the cell culture container, which is gripped by the fixing device, using the shaft portion as the axis of rotation.

10. A cell culture vessel according to claim 3, wherein the external shape is polyhedral, The above cell culture vessel is equipped with a rotating mechanism for rotating the vessel, The above-described rotation mechanism is an observation device that grips the shaft portion of the cell culture vessel and rotates the cell culture vessel using the shaft portion as the axis of rotation.

11. A microscope comprising the observation device described in claim 9 or 10.

12. A method for culturing cells or cell tissue contained in a cell culture vessel having a polyhedral shape, The above cell culture vessel is It comprises multiple compartments surrounded by a framework corresponding to each edge of a polyhedron, and these compartments are configured to hold aqueous liquids inside by surface tension, and adjacent compartments are in communication with each other. The above culture method is, The process includes accommodating a gel composition containing cells or cell tissue in the above compartment, The volume of the above compartment is 1 to 500 mm³. Culture method.

13. A method for observing cells or cell tissue contained in a cell culture vessel having a polyhedral shape, The above cell culture vessel is It comprises multiple compartments surrounded by a framework corresponding to each edge of a polyhedron, and these compartments are configured to hold aqueous liquids inside by surface tension, and adjacent compartments are in communication with each other. The above cell culture vessel is The polyhedron shape further comprises an axis portion that extends outward from any of its vertices in a direction not parallel to the normal of any of its faces, The above observation method is, The process includes rotating the cell culture vessel using the shaft portion of the cell culture vessel as the axis of rotation, The volume of the above compartment is 1 to 500 mm³. Observation method.

14. A method for observing cells or cell tissue contained in a cell culture vessel having a polyhedral shape, The above cell culture vessel is It comprises multiple compartments surrounded by a framework corresponding to each edge of a polyhedron, and these compartments are configured to hold aqueous liquids inside by surface tension, and adjacent compartments are in communication with each other. The fixing device for the above cell culture vessel is, A gripping part for gripping the cell culture vessel, The device comprises a shaft portion connected to the gripping portion, which extends outward from any of the vertices of the polyhedron shape in a direction not parallel to the normal of any of the faces of the polyhedron shape, The above observation method is, The process includes gripping the cell culture vessel with the above-mentioned fixing device and rotating the cell culture vessel using the above-mentioned shaft as the axis of rotation, The volume of the above compartment is 1 to 500 mm³. Observation method.

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