Method for controlling cell arrangement

Abstract

To provide a method for controlling cell arrangement that increases positional accuracy of droplets and enhances self-aggregability of cells when forming a spheroid in which three or more droplets are connected.SOLUTION: A substrate for supplying cells to be cultured is prepared. A first coating liquid application step for applying a first coating liquid (A) containing a first solvent onto the substrate is performed one or more times. A second coating liquid (B) is dropped so as to cover the first coating liquid (A). Cells are included in at least one application among the one or more times of application of the first coating liquid (A) in the first coating liquid application step. The substrate is a low cell adhesion substrate.SELECTED DRAWING: Figure 16

Description

[Technical Field]

[0001] The present invention relates to a method for controlling cell placement. [Background technology]

[0002] In recent years, organoid research has shown that controlling the placement of cells, proteins, and additional factors within a tissue is important. One method for achieving this is to place two types of tissue in close proximity to each other, creating a single tissue. Another method involves applying chemical, physical, and electrical stimuli from one direction to a single tissue to induce differentiation within the tissue.

[0003] In the following Non-Patent Document 1, organoids of the foregut and hindgut were prepared, and then the organoids of the foregut and midgut were brought into close proximity to form a single cellular tissue. Non-Patent Document 1 also reports that by further culturing, the liver region, gallbladder region, and pancreas region develop from the boundary between the foregut and midgut.

[0004] In Japanese Patent No. 7142840 (Patent Document 1), a water-repellent substrate is prepared in the gas phase. First and second hydrogel droplets are gelled on the substrate while connected to each other, thereby forming a cell-containing hydrogel body. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Patent No. 7142840 [Non-patent literature]

[0006] [Non-Patent Document 1] Hiroyuki Koike et al., "Modelling human hepato-biliary-pancreatic organogenesis from the foregut-midgut boundary", Nature 574, p.112-116 (2019) Summary of the Invention [Problem to be solved by the invention]

[0007] Non-Patent Document 1 discloses a technique for bringing two hydrogels containing cell tissues into close proximity to each other. However, Non-Patent Document 1 suggests that it would be difficult to bring three or more hydrogels into close proximity to each other.

[0008] On the other hand, Patent Document 1 suggests that it is possible to construct a cellular tissue (spheroid) in which three or more hydrogel droplets are connected by connecting three or more hydrogel droplets. However, Patent Document 1 does not consider placement control, such as the position of the droplets or the order in which the droplets are supplied. Patent Document 1 also does not consider the substrate onto which the droplets are supplied. For this reason, when forming a cellular tissue (spheroid) in which three or more droplets are connected, Patent Document 1 may have difficulty improving the positional accuracy of each droplet and increasing the cohesion between cells. Therefore, Patent Document 1 leaves room for improvement in terms of the positional accuracy of each droplet and the cohesion between cells.

[0009] The present invention has been made in view of the above-mentioned problems, and an object of the present invention is to provide a method for controlling cell arrangement that can improve the positional accuracy of droplets and enhance the self-aggregation of cells when forming spheroids in which three or more droplets are connected. [Means for solving the problem]

[0010] In a method for controlling cell placement according to the present disclosure, a substrate for supplying cells to be cultured is prepared. A first coating liquid application step is performed in which a first coating liquid containing a first solvent is applied onto the substrate at least once. A second coating liquid dripping step is performed in which a second coating liquid containing a thickener and a second solvent is dripped onto the substrate so as to cover the first coating liquid. At least one of the applications of the first coating liquid in the first coating liquid application step contains the cells. The substrate is a low cell-adhesion substrate. [Effects of the Invention]

[0011] The above control method includes a step of supplying a first coating liquid and a second coating liquid covering the first coating liquid, and the substrate is a low cell-adhesion substrate. This provides a method for controlling cell arrangement that improves droplet positioning accuracy and enhances cell self-aggregation when forming spheroids consisting of three or more connected droplets. [Brief explanation of the drawings]

[0012] [Figure 1] 1 is a schematic front view illustrating an example of a coating device according to an embodiment. [Figure 2] 2 is a schematic diagram showing a needle coating mechanism of the coating device shown in FIG. 1. FIG. [Figure 3] FIG. 1 is a schematic perspective view of a plate serving as a low cell adhesion substrate. [Figure 4] FIG. 2 is a schematic cross-sectional view showing a first example of the shape of a well formed in a plate. [Figure 5] FIG. 10 is a schematic cross-sectional view showing a second example of the shape of a well formed in a plate. [Figure 6] FIG. 10 is a schematic cross-sectional view showing a third example of the shape of a well formed in a plate. [Figure 7] FIG. 1 is a schematic diagram of a dish. [Figure 8] FIG. 2 is a schematic cross-sectional view of the bottom surface of the dish. [Figure 9] FIG. 3 is a schematic diagram showing the positional relationship between two first application liquids. [Figure 10] FIG. 3 is a schematic diagram showing an example of the positional relationship between a first coating liquid and a second coating liquid. [Figure 11]FIG. 2 is a schematic diagram showing a state before a first application liquid is applied in the embodiment. [Figure 12] FIG. 3 is a schematic diagram showing the composition of a first coating liquid in the embodiment. [Figure 13] FIG. 3 is a schematic diagram showing a step of applying a first application liquid in the embodiment. [Figure 14] FIG. 3 is a schematic diagram showing a state after a first coating liquid has been applied in the embodiment. [Figure 15] FIG. 10 is a schematic view showing a step of supplying a second coating liquid in the embodiment. [Figure 16] FIG. 16 is a schematic diagram showing the state inside the well after the step of FIG. 15 has been performed. [Figure 17] FIG. 1 is a schematic diagram showing a step of supplying a culture medium in an embodiment. [Figure 18] FIG. 2 is a schematic diagram showing the state of cells in a first application liquid before culture. [Figure 19] FIG. 2 is a schematic diagram showing the state of cells in a first application liquid after culture. [Figure 20] 1 shows phase contrast microscope images taken immediately after application of the application liquid and after culture in Example 1. [Figure 21] 10 is a schematic diagram showing the application state of the first coating liquid A1 and the first coating liquid A2 in Example 2. FIG. [Figure 22] 1 shows phase contrast microscope images and fluorescence microscope images taken immediately after application of the application liquid and after culture in Example 2. [Figure 23] 10 is a schematic diagram showing the application state of the first coating liquid A1 and the first coating liquid A2 in Example 3. FIG. [Figure 24] 1 shows phase contrast microscope images and fluorescence microscope images taken immediately after application of the application liquid and after culture in Example 3. DETAILED DESCRIPTION OF THE INVENTION

[0013] (Introduction) First, a brief description of the cell arrangement control method according to this embodiment will be given. As shown in FIG. 16, in the cell arrangement control method according to this embodiment, a substrate (plate 8: see FIG. 3) for supplying cells to be cultured is first prepared. A first coating liquid application step is performed in which a first coating liquid A containing a first solvent is applied to the substrate at least once. A second coating liquid dropping step is performed in which a second coating liquid B containing a thickener and a second solvent is dropped so as to cover the first coating liquid A. The cells are included in at least one of the applications of the first coating liquid A in the first coating liquid application step. The substrate is a low-cell-adhesion substrate. That is, the substrate has properties that make it difficult for cells contained in the coating liquid to adhere to it. More specifically, a low-cell-adhesion substrate refers to a substrate whose surface has a contact angle of water (pure water) that is greater than 60° and less than 70°. That is, a low-cell-adhesion substrate has a contact angle of water (pure water) that is greater than 0° and less than 60°, or greater than 70° and less than 180°.

[0014] In this specification, the term "cellular tissue" refers to one unit (one piece) of cell-containing coating liquid (bioink) released by one application of the first coating liquid from the coating device 100 (cell aggregation is not taken into consideration). In this specification, the term "spheroid" refers to a cell tissue in which multiple cells, for example, 1,000 or more cells, aggregate and are organized into a single spherical or nearly spherical mass through three-dimensional culture. In other words, in this embodiment, a spheroid is formed when, for example, three or more cellular tissues are connected to each other and the cells contained in the cellular tissues aggregate.

[0015] (Embodiment) (Device configuration) FIG. 1 is a schematic front view showing an example of a coating apparatus according to an embodiment. For convenience of explanation, the X, Y, and Z directions are introduced. As shown in FIG. 1, the coating apparatus 100 includes a needle coating mechanism 104 and a dripping mechanism 105 as a coating mechanism 107 capable of supplying the coating material to be coated. In this specification, "coating" may include both supplying the coating material (coating liquid) using a coating needle (described later) and supplying the coating material by dripping. Therefore, the former supply of the coating liquid using a coating needle may be referred to as "needle coating," particularly in the description of FIG. 1. The coating apparatus 100 in FIG. 1 includes two needle coating mechanisms 104 and two dripping mechanisms 105. The two needle coating mechanisms 104 are needle coating mechanism 104-1 and needle coating mechanism 104-2, and are arranged at an interval in the X direction. The two dripping mechanisms 105 (dispensers) are dripping mechanism 105-1 and dripping mechanism 105-2, and are arranged side by side with a gap in the X direction. Furthermore, the needle application mechanism 104 and dripping mechanism 105 are arranged side by side with a gap in the X direction.

[0016] The X-axis stage 101 is movable along the X direction, which is the horizontal direction. The Y-axis stage 102 is movable along the Y direction, which is the horizontal direction. Specifically, for example, a guide unit is installed on the underside of the X-axis stage 101 or the Y-axis stage 102. The guide unit is slidably connected to a guide rail (not shown). For example, the upper surface of the X-axis stage 101 serves as a mounting surface on which a plate 8 can be placed. In FIG. 1, the X-axis stage 101 is placed on the Y-axis stage 102, and the plate 8 is placed on the X-axis stage 101. However, conversely, the X-axis stage 101 may be placed on the Y-axis stage 102, and the plate 8 may be placed on the X-axis stage 101.

[0017] The needle coating mechanism 104, the dripping mechanism 105, and the observation optical system 106 are connected to a member movable in the Z direction, such as a Z-axis table. In other words, the needle coating mechanism 104, the dripping mechanism 105, and the observation optical system 106 are held within the coating device 100 so that they can move in the Z direction. The observation optical system 106 observes and measures the position on the plate 8 where the coating material is to be applied. The observation optical system 106 may be equipped with a CCD camera that converts the observed image into an electrical signal. The observation optical system 106 may observe the plate 8 using visible light. However, the observation of the plate 8 is not limited to visible light, and may also be performed using infrared light, X-rays, ultrasound, or the like. Depending on the material of the plate 8, the plate 8 may also be observed using magnetism. The plate 8 observed using means other than visible light does not need to be transparent or translucent and may be opaque.

[0018] FIG. 2 is a schematic diagram showing the needle coating mechanism of the coating device shown in FIG. 1. As shown in FIG. 2, the needle coating mechanism 104 of this embodiment mainly includes a servo motor 41, a cam 43, a bearing 44 held in contact with the cam surface of the cam 43, a cam connecting plate 45, a movable part 46, a movable base 35 holding the coating needle holder 20, and a coating material container 21. The configuration of the needle coating mechanism 104 shown in FIG. 2 is common to both the needle coating mechanism 104-1 and the needle coating mechanism 104-2 shown in FIG. 1. The coating needle holder 20 is detachable from the movable base 35. In other words, the movable base 35, which serves as a base body, detachably holds the coating needle holder 20.

[0019] In the needle coating mechanism 104, the servo motor 41 is installed so that its central axis extends in the Z-axis direction shown in FIG. 1 . A cam 43 is connected to the rotation shaft of the servo motor 41. The cam 43 is rotatable around the central axis of the servo motor 41. The cam 43 includes a center portion connected to the rotation shaft of the servo motor 41 and a flange portion connected to one end of the center portion. The upper surface of the flange portion (the surface facing the servo motor 41) is a cam surface. This cam surface is formed in an annular shape along the outer periphery of the center portion and is formed in a sloped shape so that the distance from the bottom surface of the flange portion varies. Specifically, the cam surface includes an upper end flat region that is farthest from the bottom surface (thickest), a lower end flat region that is spaced apart from the upper end flat region, and a sloped portion that smoothly connects the upper end flat region and the lower end flat region. The lower end flat region is the region that is closest to the bottom surface (thinnest).

[0020] A bearing 44 is arranged so as to contact the cam surface of this cam 43. A cam connecting plate 45 is connected to this bearing 44. The other end of the cam connecting plate 45, opposite to one end connected to the bearing 44, is fixed to a movable part 46. A movable base 35 serving as a base body is connected to this movable part 46. A coating needle holder 20 is installed on this movable base 35. The coating needle holder 20 includes a coating needle 24. The coating needle 24 is capable of applying a coating material to, for example, a well 9A of the plate 8. The coating needle 24 is arranged so as to protrude from the coating needle holder 20 on the underside of the coating needle holder 20 (the lower side opposite the side on which the servo motor 41 is located). A coating material container 21 is arranged below the coating needle holder 20. The coating needle 24 is held in an inserted state in the coating material container 21.

[0021] A fixed pin is fixed to the movable part 46. The other fixed pin is fixed to the stand that holds the servo motor 41. A spring is installed to connect these fixed pins. This spring causes the movable part 46 to receive a force toward the coating material container 21. The force of this spring also keeps the bearing 44 pressed against the cam surface of the cam 43.

[0022] Furthermore, the movable part 46 and the movable base 35 are connected to a linear guide installed on a stand that holds the servo motor 41, and are movable along the Z-axis direction.

[0023] In the needle coating mechanism 104 described above, the servo motor 41 is driven to rotate the rotation shaft of the servo motor 41, thereby rotating the cam 43. As a result, the position of the bearing 44, which is in contact with the cam surface of the cam 43, in the Z-axis direction varies in accordance with the rotation of the rotation shaft of the servo motor 41. The movable part 46 and the movable base 35 then move in the Z-axis direction in response to the positional variation of the bearing 44 in the Z-axis direction, thereby changing the position of the coating needle 24 in the Z-axis direction. In other words, the coating needle 24 can be reciprocated in the Z-axis direction. As a result of this movement, when the coating needle 24 is positioned upward in the Z-axis direction, the tip of the coating needle 24 is immersed in the coating material container 21, which contains the liquid material. In this state, the coating needle 24 projects downward from a tip hole in the bottom of the coating material container 21, thereby performing a coating operation. With the liquid material attached to the tip of the coating needle 24, the tip of the coating needle 24 projects from the tip hole of the coating material container 21 and exits the coating material container 21. At this time, the liquid material is pulled upward by surface tension, and a substantially constant amount of the liquid material adheres to the tip of the application needle 24. The liquid material thus adhered is transferred to the inside of the well 9A of the plate 8, thereby achieving a highly reproducible application process.

[0024] FIG. 3 is a schematic perspective view of a plate serving as a low cell-adhesion substrate. As shown in FIG. 3, in this embodiment, the coating liquid as a liquid material is synonymous with the above-mentioned "droplet." Furthermore, the coating liquid is synonymous with "bioink." The coating liquid is applied and supplied to the inside of multiple wells 9A (recesses) formed in a plate 8 (low cell-adhesion substrate). The plate 8 has a thickness in the Z-axis direction and is formed by a plate main body 8A. Multiple wells 9A are formed on the uppermost surface of the plate main body 8A. The multiple wells 9A are recessed portions of the upper surface of the plate 8 (plate main body 8A). The multiple wells 9A may be formed at intervals from one another, for example, in eight rows in the Y-axis direction and twelve rows in the X-axis direction in FIG. 3, for a total of 96 wells. When viewed from above in the Z direction, the wells 9A may have any planar shape, such as a circle, a rectangle, or a square. Furthermore, the number of wells 9A formed in the plate 8 is not limited to the above-mentioned 96. The number of wells 9A in the plate 8 may be any of 6, 12, 24, 48, 96, 384, and 1536. Typically, the maximum dimension of the wells 9A when viewed from above exceeds 1 mm. Here, the maximum dimension refers to the value of the largest part of the dimensions of the wells 9A when viewed from above. For example, if the wells 9A are oval, the maximum dimension is the dimension along the long axis.

[0025] Fig. 4 is a schematic cross-sectional view showing a first example of the shape of wells formed in a plate. As shown in Fig. 4, a plurality of wells 9A formed on the surface of plate 8 (plate body 8A) may be formed to have wall surfaces 91 extending in a direction intersecting the surface (top surface 90) and bottom surfaces 92 extending along the surface (top surface 90). The bottom surface 92 is a flat surface that is located at a position farthest from the top surface 90 of each well 9A. The wells 9A are formed by the bottom surface 92 and wall surfaces 91 that are continuous with the outer edge of the bottom surface 92 and extend so as to intersect with the bottom surface 92.

[0026] In the well 9A in FIG. 4, the boundary between the wall surface 91 and the bottom surface 92 forms a ridge like the intersection of two planes. In FIG. 4, the wall surface 91 is slightly inclined relative to a direction perpendicular to the surface (top surface 90). Therefore, the area of ​​the bottom surface 92 is smaller than the area of ​​the opening in the top surface 90 that is removed by the well 9A in a plan view. This configuration is also acceptable. However, as another example, the wall surface 91 may be perpendicular to the top surface 90. In other words, in the cross section of FIG. 4, the wall surface 91 may be perpendicular to the top surface 90, or may be inclined and not perpendicular to the top surface 90. The cross section in FIG. 4 refers to a cross section (plane) extending along a straight line extending in the vertical direction, and this also applies to the following FIGS. 5 and 6. The wall surface 91 may be shaped like a portion of the side surface of a cylinder or a cone. Alternatively, the wall surface 91 may be shaped like a portion of the side surface of a prism or a pyramid.

[0027] FIG. 5 is a schematic cross-sectional view showing a second example of the shape of a well formed in a plate. As shown in FIG. 5, a well 9A formed in a plate body 8A has a wall surface 91 and a bottom surface 92. The wall surface 91 is the same as that of the well 9A in FIG. 4. The bottom surface 92 is arranged so as to be continuous with the lowermost part of the wall surface 91. The well 9A in FIG. 5 has a curved bottom surface 92 (the bottom part farthest from the uppermost surface 90). The curved surface of the bottom surface 92 may be a part of a sphere or a part of the surface of an ellipsoid. In FIG. 5, the boundary between the planar wall surface 91 and the bottom surface 92 is a rounded curved surface in the cross section of FIG. 5. This boundary portion may also be a part of a sphere or a part of the surface of an ellipsoid. As a result, the shape of the bottom surface 92 in the cross section of FIG. 5 is U-shaped.

[0028] FIG. 6 is a schematic cross-sectional view showing a third example of the shape of a well formed in a plate. As shown in FIG. 6, a well 9A formed in a plate body 8A has a wall surface 91 and a bottom surface 92. The wall surface 91 is similar to the well 9A in FIG. 4. In the cross section of FIG. 6, both the wall surface 91 and the bottom surface 92 are inclined with respect to the vertical direction perpendicular to the top surface 90. In the well 9A formed in the plate body 8A, the bottom surface 92 (the bottom part farthest from the top surface 90) and the wall surface 91 have different angles with respect to a line extending in the vertical direction. In FIG. 6, the boundary between the wall surface 91 and the bottom surface 92 connected thereto forms a ridge line. In the cross section of FIG. 6, the bottom surface 92 has a larger angle with respect to a line extending in the vertical direction than the wall surface 91.

[0029] The bottom surface 92 in Fig. 6 has a shape similar to the side surface of a cone or pyramid, for example. Therefore, the lowest part of the bottom surface 92 may have a pointed shape like the apex of a cone or pyramid in the cross section of Fig. 6. As a result, the shape of the bottom surface 92 in the cross section of Fig. 6 is V-shaped.

[0030] Plate 8 serving as a low cell-adhesion substrate may be a so-called multiwell plate. A multiwell plate has a plurality of wells 9A, each having a shape as shown in any one of FIGS. 4 to 6, arranged in a matrix. In other words, plate 8 in FIG. 3 is a multiwell plate. In this embodiment, spheroids may be formed from cell tissue using, for example, three or more coating solutions using a multiwell plate. In this case, spheroids may be formed in wells 9A by the coating solutions supplied into the wells 9A.

[0031] However, the plate 8 (low cell-adhesion substrate) is not limited to a multi-well plate, and may be a slide glass, a dish, or a cell desk LF.

[0032] FIG. 7 is a schematic diagram of a dish. FIG. 8 is a schematic cross-sectional view of the bottom surface of the dish. If the plate 8 is a dish as shown in FIG. 7, the coating liquid is supplied onto the bottom surface of the dish. As shown in FIG. 8, a plurality of recesses 9B are formed on the bottom surface (top surface 90) of the dish as recesses. The recesses 9B are formed on the bottom surface (top surface 90) of the dish by special microfabrication. The sizes of the recesses 9B may be random, but for example, their maximum dimension in a plan view is less than 1 mm. In other words, the recesses 9B are usually smaller than the wells 9A of a multiwell plate. Therefore, it may be possible to distinguish between the wells 9A of a multiwell plate and the recesses 9B of a dish based on their sizes. For example, the recesses 9B may be approximately circular in a plan view, have a diameter of 400 μm to 500 μm, and a depth of 100 μm to 200 μm.

[0033] 8, the coating solution is supplied to the inside of a depression 9B on the bottom surface of the dish, which may result in the formation of spheroids in the depression 9B. The depression 9B may be formed, for example, in a slide glass for spheroid formation.

[0034] If the plate 8 is a multiwell plate as shown in Figure 3, spheroids with controlled cell placement can be easily produced by simply supplying the first coating liquid A or the like directly into the multiple wells 9A formed therein. Even if the plate 8 is a so-called dish, the first coating liquid A or the like can be supplied to the bottom surface of the dish so that multiple wells are spaced apart. A dish is a circular, large, flat-shaped device with a wall around the outer edge of the bottom surface. In this way, spheroids with controlled cell placement can be easily produced when using a dish. In other words, whether the plate 8 is a multiwell plate or a dish, for example, no step of transferring droplets supplied onto the surface of the substrate into a culture vessel is required. This allows for easier supply of the coating liquid or the like compared to cases requiring such a transfer step. Furthermore, this prevents the accuracy of cell placement due to unintended movement or shape collapse of the coating liquid during droplet transfer, and the resulting decrease in the cohesion of multiple cells within the droplets.

[0035] Even when plate 8 is a low cell-adhesion substrate, the difficulty of the coating solution itself adhering to plate 8 is not an issue. When plate 8 is a low cell-adhesion substrate, cells contained in the coating solution are less likely to adhere to the surface of plate 8. From this perspective, the contact angle of water (pure water) adhering to the surface of plate 8 is preferably an angle not less than 60° and not more than 70°. In other words, the contact angle may be a small angle less than 60°, or a large angle greater than 70° and less than 180°. The numerical range of the water contact angle may vary depending on the type of cells contained in the coating solution.

[0036] First, making the contact angle of water small, less than 60°, means making the surface of plate 8, including bottom surface 92 of well 9A, the bottom surface of the dish, etc., hydrophilic. A method for achieving this will be specifically described.

[0037] From a different perspective, suppressing protein adsorption on the surface of plate 8 can make the surface hydrophilic and less susceptible to cell adhesion. Cell adhesion occurs when cell adhesive proteins added to a cell culture medium adhere to the substrate on which cells are to be cultured, and cells then adhere to the proteins. Therefore, preventing protein adhesion to the substrate can turn the substrate into a low-cell-adhesion substrate. To prevent protein adhesion to the substrate, it is preferable to reduce the free energy of the thermodynamic driving force and the free energy of the interface between the substrate and water. In other words, by making the surface of the substrate on which cells are to be cultured a nonionic hydrophilic surface, the contact angle of water on the surface is reduced, thereby suppressing protein adhesion to the surface. This can therefore suppress cell adhesion to the surface.

[0038] In the above, the substrate on which cells are to be cultured (the substrate on which proteins are attached) is the plate 8 in this embodiment, and more specifically, the bottom surface 92 of a well 9A of a multi-well plate, the top surface 90 of a dish, the surface inside a recess 9B, etc. The cell culture medium may be medium M (see FIG. 17) described below, or a medium contained as a first solvent in first coating liquid A. The cell adhesive protein added to the cell culture medium may be collagen, fibrinectin, or the like dissolved alone in a coating liquid or the like. Alternatively, the cell adhesive protein may be fetal bovine serum (FBS) or horse serum.

[0039] The recesses of the wells 9A and depressions 9B do not necessarily contribute to controlling the contact angle of water on the surface of the plate 8. The wells 9A of the multiwell plate and the depressions 9B formed on the top surface 90 of the dish serve as containers for forming spheroids. For example, one spheroid can be formed in one well 9A or one depression 9B.

[0040] Regardless of the type of plate 8, it is preferable that the plate 8 be formed using a low cell-adhesion polymer. Here, the low cell-adhesion polymer may be, for example, either an MPC polymer or P-HEMA. For example, the plate 8 may be formed by coating the surface of a substrate with either an MPC polymer or P-HEMA. MPC polymer is a biomaterial that suppresses protein adsorption. For this reason, the use of MPC polymer makes it possible to obtain a low cell-adhesion substrate.

[0041] An example of a method for forming an MPC polymer-coated plate 8 is as follows: A solution of the MPC polymer is poured onto the surface of a multiwell plate, dish, or the like, which is the plate 8. Excess solution is removed from the plate 8. The plate 8 is then dried.

[0042] Additionally, from the viewpoint of making the surface of plate 8 hydrophilic, the following can also be done. Regardless of the type of plate 8, the contact angle of water (pure water) on the surface of plate 8 may be controlled by radiation. In other words, the contact angle of water on the surface of plate 8 may be controlled by irradiating the surface with radiation. Examples of radiation that can be irradiated onto plate 8 include gamma rays. The surface of plate 8 is made hydrophilic by irradiation with radiation. As a result, plate 8 acquires properties as a non-cell-adhesive substrate.

[0043] As another method, the surface of the plate 8 (such as the wells 9A of a multi-well plate or the depressions 9B of a dish) may be irradiated with plasma. This makes the surface of the plate 8 hydrophilic, which has the effect of making it difficult for cells to adhere to the wells 9A and depressions 9B.

[0044] Second, increasing the water contact angle to a large angle exceeding 70° and approaching 180° means making the surface of plate 8, including bottom surface 92 of well 9A, the bottom surface of the dish, etc., hydrophobic. To achieve this, it is preferable to use plate 8 whose surface is coated with a hydrophobic polymer.

[0045] (Features of manufacturing method) The manufacturing method according to this embodiment, i.e., the method for controlling cell arrangement (method for manufacturing spheroids), is as follows: First, a low cell-adhesion substrate such as the multiwell plate or dish described above is prepared as plate 8 for supplying cells to be cultured.

[0046] Next, a first coating liquid containing a first solvent is applied at least once onto the plate 8. This process is called the first coating liquid application process. The first coating liquid preferably contains, in addition to the first solvent, a gel raw material, which is a material to be gelled. If the plate 8 is a multi-well plate, the first coating liquid is applied to one well 9A selected from the multiple wells 9A. If the plate 8 is a dish, the first coating liquid is applied so as to include the inside of the depression 9B of the dish. In either case, a spherical or nearly spherical droplet supplied by one application is referred to here as one coating liquid. Therefore, at least one first coating liquid is supplied by at least one application.

[0047] Note that the multiple first application liquids are considered to be one liquid that is supplied continuously in time by one needle application mechanism. First application liquids that are supplied in separate time intervals by one needle application mechanism are considered to be separate from each other. Furthermore, first application liquids that are supplied simultaneously in time by different needle application mechanisms are considered to be separate from each other.

[0048] In the first coating liquid application step, the first coating liquid is applied at least once (once or twice or more). In the first coating liquid application step, at least one (one or two or more) first coating liquid is applied. In at least one application of the first coating liquid in the first coating liquid application step, the cells to be cultured are contained in the first coating liquid. When two or more first coating liquids are applied, for example, the first coating liquid applied the first time contains one or more types of cells. However, when two or more first coating liquids are applied, the first coating liquid applied the second or subsequent times may not contain cells. The first coating liquid applied the second or subsequent times may also contain cells. Alternatively, the first coating liquid applied the first time may not contain cells, and the first coating liquid applied any one of the second or subsequent times may contain cells.

[0049] In the step of applying the first coating liquid onto plate 8, N copies of the first coating liquid (N is a natural number of 2 or more) are applied. In particular, in the step of applying the first coating liquid onto plate 8, the first coating liquid may be applied three or more times onto plate 8 (e.g., into well 9A) and arranged on plate 8 (e.g., into well 9A). In particular, in the above step, the first coating liquid may be applied four or more times onto plate 8 (e.g., into well 9A) and arranged on plate 8 (e.g., into well 9A).

[0050] 9 is a schematic diagram showing the arrangement relationship of two first coating liquids. As shown in FIG. 9, the dimension of the first coating liquid A is defined as the first dimension r 1n (1≦n≦N). That is, the first dimension of the first of N first coating liquids A is r 11 The first dimension of the second piece is r 12 In FIG. 9, the dimensions of two arbitrarily selected first liquids A from N (N is a natural number of 2 or more) are expressed as r 1n , r 1(n+1) (n is a natural number in the range of 1≦n≦N-1). The distance between the centers (centroids) of the two first liquids A is defined as d n,n+1In this case, the positional relationship of the two first coating liquids may be such that they do not overlap each other as in (A) in Figure 9, such that one is completely contained within the other as in (B), or such that they partially overlap as in (C). (A) includes a case where the two are circumscribing each other, and (B) includes a case where one is inscribed within the other.

[0051] In the case of (A) in FIG. 9, the following formula (1) holds, in the case of (B) the following formula (2) holds, and in the case of (C) the following formula (3) holds. The equal sign in formula (1) holds when the two first coating liquids A are in contact with each other externally in FIG. 9 (A). The equal sign in formula (2) holds when one first coating liquid A is in contact with the other internally in FIG. 9 (B). In formula (3), r 1n =r 1(n+1) If so, d n,n+1 <r 1n +r 1(n+1) Only holds.

[0052] (Number 1)d n,n+1 ≧r 1n +r 1(n+1) ···(1) (Number 2)d n,n+1 ≦r 1(n+1) -r 1n ···(2) (number 3)r 1(n+1) -r 1n <d n,n+1 <r 1n +r 1(n+1) ···(3) In this embodiment, the first coating liquid A is applied to a position where either the above formula (2) or formula (3) is satisfied, thereby forming spheroids in which multiple first coating liquids A are linked together.

[0053] When N first coating liquids (N is a natural number of 2 or more) are applied, the components of each of the N first coating liquids (for example, the type of first solvent contained therein) may be the same. However, the components of each of the N first coating liquids may be different from one another. Some of the components of the N first coating liquids may be the same, and the other components may be different from the above-mentioned parts. In either case, the effect of this embodiment can be obtained.

[0054] When N first coating liquids (N is a natural number of 2 or greater) are coated, the types of cells contained in at least two of the N first coating liquids may be different from each other. Alternatively, the types of cells contained in all N first coating liquids may be the same. Alternatively, the types of cells contained in some of the N first coating liquids may be the same, and the types of cells contained in the other parts may be different from the types of cells contained in the above-mentioned parts. In any of these cases, the effects of this embodiment can be obtained.

[0055] The step of applying the first application liquid uses a needle-type bioprinter. That is, the first application liquid is applied, for example, by the needle application mechanism 104 (needle application mechanisms 104-1 and 104-2) in FIG. 1. This allows the first application liquid to be applied in minute amounts, i.e., to very small dimensions, into well 9A, etc. Furthermore, the needle-type application method allows application of the first application liquid even if it is highly viscous. However, the first application liquid may also be applied (supplied) by a method other than the needle-type application method. The step of applying the first application liquid may use any one selected from the group consisting of a needle-type bioprinter, a dispenser (such as the dripping mechanism 105 (see FIG. 1)), an inkjet, and a pipette. Each of these methods allows the first application liquid to be stably supplied to the desired position.

[0056] The second coating liquid is dripped (applied) so as to cover the first coating liquid A. This process is called the second coating liquid dripping process. The second coating liquid contains a thickener and a second solvent. In addition to the above, various additives may be added to the second coating liquid. FIG. 10 is a schematic diagram showing an example of the positional relationship between the first coating liquid and the second coating liquid. As shown in FIG. 10, in this embodiment, the first coating liquid A is arranged so that it is neither inscribed nor circumscribed in the second coating liquid B. The second coating liquid B may be dripped multiple times to form a single second coating liquid B. In FIG. 10, as an example, the second coating liquid B is supplied (for example, into well 9A) so as to cover two first coating liquids A1 and A2 arranged as shown in FIG. 9C. However, the second coating liquid B may also be supplied (for example, into well 9A) so as to cover two first coating liquids A1 and A2 arranged as shown in FIG. 9B.

[0057] The step of dropping the second coating liquid B is performed by a dispenser. That is, the second coating liquid B is dropped by, for example, the dropping mechanism 105 (dropping mechanism 105-1) in Fig. 1. However, the method is not limited to this. In addition to the above, the step of applying the second coating liquid B may also use any one selected from the group consisting of a coating needle method, an inkjet method, and a pipette (manual).

[0058] After supplying the second coating liquid B, for example, by dropping a medium (medium M described next) onto the first coating liquid A and the second coating liquid B in the well 9A, spheroids in which the cell tissues are connected are formed.

[0059] The step of dripping the medium is performed by a dispenser. That is, the medium is dripped by, for example, drip mechanism 105 (dripping mechanism 105-2) in FIG. 1 . However, this is not limiting. In addition to the above, the step of applying the medium may also use any one selected from the group consisting of an application needle method, an inkjet method, a dispenser, a micropump, and a pipette (manual).

[0060] (Explanation of the specific manufacturing method using diagrams) Here, an example using a multiwell plate is illustrated. However, similar procedures apply when using equipment other than a multiwell plate, such as a dish or a glass slide. FIG. 11 is a schematic diagram showing the state before the first coating liquid is applied in an embodiment. As shown in FIG. 11, the tip of the applicator needle 24 constituting the bioprinter is first immersed in the first coating liquid A (first bioink), and the first coating liquid A is applied to the tip of the applicator needle 24. FIG. 12 is a schematic diagram showing the composition of the first coating liquid in an embodiment. As shown in FIG. 12, the first coating liquid A is obtained by mixing the cells C to be cultured, collagen as a gelling agent (gel raw material), and the first solvent m. The cells C in FIG. 12 include C1 and C2, which will be described later. FIG. 13 is a schematic diagram showing the process of applying the first coating liquid in an embodiment. As shown in FIG. 13, the tip of the applicator needle 24 to which the first coating liquid A is applied comes into contact with, for example, the bottom surface 92 (see FIG. 4) of the well 9A (see FIG. 4). This is achieved by moving the application needle 24, to which the first application liquid A has been attached, downward as shown by arrow M1 in FIG. 11. As a result, the first application liquid A is applied to the bottom surface 92 of the well 9A. FIG. 14 is a schematic diagram showing the state after the first application liquid has been applied in this embodiment. As shown in FIG. 14, the application needle 24 then moves upward as shown by arrow M2. In this way, the first application liquid A is applied to a container such as the well 9A using a bioprinter of the so-called pin type (a needle type such as the needle application mechanism 104 in FIG. 1).

[0061] FIG. 15 is a schematic diagram showing a step of supplying a second coating liquid in an embodiment. FIG. 16 is a schematic diagram showing the state inside a well after the step of FIG. 15 has been performed. As shown in FIGS. 15 and 16, a second coating liquid B (second bioink) is supplied into well 9A so as to cover the first coating liquid A applied to the inside of well 9A. The second coating liquid B may be dripped using a dispenser, for example, but the method of supplying the second coating liquid B is not limited to this. The second coating liquid B may be supplied by any method selected from the group consisting of a pin method, an inkjet method, a dispenser method (a method using dripping mechanism 105 of FIG. 1), and manual delivery using a pipette.

[0062] 17 is a schematic diagram showing a step of supplying a culture medium in an embodiment. As shown in FIG. 17, after the step of supplying the second coating liquid B, the culture medium M is supplied into the well 9A. The culture medium M is supplied so as to be immersed in and cover the first coating liquid A and the second coating liquid B. The method of dropping the culture medium M is not particularly limited. The culture medium M may be supplied by any method selected from the group consisting of a pin method, an inkjet method, a dispenser method, and manual supply using a pipette. Alternatively, the culture medium M may be supplied by a dispenser or a micropump.

[0063] FIG. 18 is a schematic diagram showing the state of cells in the first coating liquid before culture. FIG. 19 is a schematic diagram showing the state of cells in the first coating liquid after culture. As shown in FIGS. 18 and 19 (and FIG. 12), cells C in the first coating liquid A before culture contain cardiomyocytes C1 and cardiac fibroblasts C2. The proportion of cardiomyocytes C1 in the cells C is 75% or more, preferably 80% or more. The proportion of cardiac fibroblasts C2 in the cells C is 20% or less. Therefore, when the proportion of cardiomyocytes C1 is lowest, C1:C2 = 80:20, and when the proportion of cardiomyocytes C1 is highest, C1:C2 = 100:0. In other words, in the above proportional formula, the proportion (%) of cells C is 80≦C1≦100 (or 75≦C1≦100) and 0≦C2≦20. The cell volume concentration of the cardiomyocytes C1 and cardiac fibroblasts C2 contained in the first coating solution A is preferably 0.001 vol% or more and 50 vol% or less. Of these, the cell volume concentration is more preferably 1 vol% or more and 30 vol% or less. Of these, the cell volume concentration is most preferably 25 vol%. By setting the ratio of the number of cardiomyocytes C1 and cardiac fibroblasts C2 and the cell volume concentration as described above, cellular tissue can be stably formed by culturing the cells, as in the Examples described below. Through culturing, both the cardiomyocytes C1 and cardiac fibroblasts C2 grow from the state shown in FIG. 18 to the state shown in FIG. 19, and myocardial tissue is formed.

[0064] (material) The first coating solution A is composed of the cells to be cultured, a gel (a gel raw material if liquid) for producing spheroids, and a first solvent. In addition to the above, various additives may be added to the first coating solution A.

[0065] The type of cell is not particularly limited. Normal cells or cells derived from various diseases may be used. Alternatively, cells that have undergone gene transfer, genetic modification, or genetic recombination may be used. Cells may be derived from any animal (animal origin) including humans, mice, rats, and monkeys. Cells may be differentiated cells derived from stem cells. In other words, cells may be differentiated cells derived from iPS cells or ES cells. Cells may be mesenchymal stem cells. Cells may be primary cells or established cell lines.

[0066] The cell types may be any of neurons, cardiomyocytes, fibroblasts, vascular endothelial cells, hepatocytes, Kupffer cells, hepatic stellate cells, pit cells, epithelial cells, and skeletal muscle cells, i.e., cells derived from various organs. Neurons include central neurons, sympathetic neurons, parasympathetic neurons, sensory neurons, interneurons, motor neurons, microglia, astrocytes, oligodendrocytes, ependymal cells, Schwann cells, and satellite cells. Cardiomyocytes include ventricular myocytes and atrial myocytes.

[0067] The density of the cells in the first coating liquid A is not particularly limited. However, the density of the cells is, for example, 1×10 2 cells / mL or more 1×10 9 It may be less than cells / mL.

[0068] First Coating Liquid A does not necessarily have to contain a gel (a gel raw material before solidification: hereinafter referred to as a gel). For example, First Coating Liquid A may contain either a natural polymer or a synthetic polymer. When a gel is used in First Coating Liquid A, the material of the gel is not particularly limited. The gel in First Coating Liquid A may be, for example, any of collagen, fibrin, Matrigel, gelatin, sodium alginate, gelatin methacryloyl (GelMA), cellulose, cellulose nanofiber, chitin, chitosan, chitin nanofiber, chitosan nanofiber methylcellulose, carboxymethylcellulose, hydroxybutylcellulose, sodium alginate, sodium hyaluronate, polyethylene glycol, gellan gum, carrageenan, pectin, xanthan gum, gelatin, agarose, and polyvinyl alcohol.

[0069] The material of the first solvent is not particularly limited. However, the first solvent may be the same material as the culture medium or a buffer solution. For example, the first solvent may be any of DMEM, DMEM / Ham F-12, αMEM, RPMI-1640, Williams' medium, M199, commercially available culture media for various cells, PBS solution (+ or -), Tris buffer, and Tyrode's buffer.

[0070] The materials of the various additives in the first coating liquid are not particularly limited. The various additives may be drugs acting on cells, cell growth factors, cytokines, hormones, transcription factors, ECM, proteins, antibodies, thickeners, salts, etc. The various additives may be low-molecular-weight compounds, medium-molecular-weight compounds, or high-molecular-weight compounds. For example, the additives include T3, T4, IGF (insulin-like growth factor: IGF-I), epidermal growth factor (EGF), TGF, basic fibroblast growth factor (bFGF), fibroblast growth factor (FGF2), vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), granulocyte-colony stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), platelet-derived growth factor (PDGF), EPO, TPO, hepatocyte growth factor (HGF), insulin, dexamethasone, isoproterenol, B27 (registered trademark) supplement, N2 supplement, fetal bovine serum (FBS), collagen, fibrin, Matrigel, gelatin, fibronectin, vitronectin, laminin, proteoglycan, nidogen, ROCK The inhibitor may be any of sodium alginate, gelatin methacryloyl (GelMA), cellulose, cellulose nanofiber, chitin, chitosan, chitin nanofiber, chitosan nanofiber, methylcellulose, carboxymethylcellulose, hydroxybutylcellulose, sodium alginate, sodium hyaluronate, polyethylene glycol, gellan gum, carrageenan, pectin, xanthan gum, gelatin, agarose, and polyvinyl alcohol.

[0071] There are no particular restrictions on the type of thickener contained in the second coating liquid B. For example, the thickener may be any one of cellulose, cellulose nanofiber, chitin, chitosan, chitin nanofiber, chitosan nanofiber, methylcellulose, carboxymethylcellulose, hydroxybutylcellulose, sodium alginate, sodium hyaluronate, polyethylene glycol, gellan gum, carrageenan, pectin, xanthan gum, gelatin, agarose, and polyvinyl alcohol.

[0072] The material of the second solvent is not particularly limited, but the same type as the material of the first solvent can be used. The various additives in the second coating liquid are not particularly limited, but the same types as the various additives in the first coating liquid can be used.

[0073] The medium for culturing spheroids, i.e., medium M (see FIG. 15 ), which is applied after second coating liquid B in the production process, is not particularly limited in material. However, medium M may be, for example, DMEM, DMEM / Ham F-12, αMEM, RPMI-1640, Williams' medium, M199, or any of commercially available media specifically designed for various cells. These various types of medium M may be mixed in any ratio. Various additives may also be added to medium M. The materials of the various additives to medium M may be the same as those of the various additives to first coating liquid A or second coating liquid B described above.

[0074] (Action and effect) In the method for controlling cell arrangement (method for producing spheroids) according to this embodiment, a substrate (plate 8) is prepared for supplying cells C to be cultured. A first coating liquid application step is performed in which a first coating liquid A containing a first solvent m is applied onto the plate 8 at least once. A second coating liquid dropping step is performed in which a second coating liquid B containing a thickener and a second solvent is dropped so as to cover the first coating liquid A. Cells C are included in at least one application of first coating liquid A in the first coating liquid application step. The plate 8 is a low cell-adhesion substrate.

[0075] In this embodiment, the second coating liquid B containing a thickener and a second solvent is dropped so as to cover the first coating liquid A. This makes it easier to control the position of the first coating liquid A so that it is fixed. This also makes it easier to form spheroids in which the cells self-aggregate. By covering the first coating liquid A with the second coating liquid B, the drying of the first coating liquid A can be suppressed. This makes it possible to suppress the death of cells in the first coating liquid A.

[0076] A low-cell-adhesion substrate is used as the substrate (plate 8). Therefore, after coating the substrate with the first coating liquid A, etc., the cells contained in the first coating liquid A are less likely to adhere to the surface of the substrate. If the cells in the first coating liquid A adhere and spread on the substrate, the cells C (see FIG. 12) are less likely to aggregate, and the position of the coating liquid constituting the spheroid is shifted. This makes it difficult to produce spheroids with high positional accuracy of the coating liquid and cells C, and high cohesion between the cells C in the coating liquid. To solve this problem, the present embodiment suppresses cell adhesion to the substrate. As a result, even when three or more first coating liquids A are formed, spheroids can be produced with high positional accuracy of the first coating liquid A and cells C, and high cohesion between the cells C in the first coating liquid A. By increasing the positional accuracy of multiple first coating liquids A, the positions of cells, proteins, added factors, etc. can be controlled with high precision.

[0077] In the above-described method for controlling cell arrangement, in the step of applying the first coating liquid A onto the plate 8, the first coating liquid A is applied three or more times onto the plate 8 and arranged on the plate 8. In other words, according to the above-described technique of the present embodiment, when spheroids are formed by linking together droplets (bioink) of the first coating liquid A that have been applied three or more times, the positional accuracy of the droplets can be improved, and the self-aggregation of the cells C in the first coating liquid A can be enhanced.

[0078] In the above-described method for controlling cell positioning, a low-cell-adhesion substrate may be formed by coating the surface of the substrate with either MPC polymer or P-HEMA. This makes it possible to hydrophilize the surface of plate 8 (the bottom surface of well 9A, the surface of depression 9B, etc.), turning plate 8 into a non-cell-adhesion substrate. As a result, as described above, when spheroids are formed by joining three or more applied droplets (bioink) of First Coating Liquid A, the positional accuracy of the droplets and cells can be improved, and the self-aggregation of cells C in First Coating Liquid A can be enhanced.

[0079] The above-described method for controlling cell arrangement may further include a step of controlling the contact angle of the surface with water by irradiating the surface of plate 8 with radiation. This makes the surface of plate 8 (the bottom surface of well 9A, the surface of depression 9B, etc.) hydrophilic, and plate 8 can be made into a cell non-adhesive substrate. Therefore, as described above, when spheroids are formed by joining three or more applied droplets (bioink) of First Coating Liquid A, the positional accuracy of the droplets and cells can be improved, and the self-aggregation of cells C in First Coating Liquid A can be enhanced.

[0080] In the above-described method for controlling cell arrangement, a plurality of recesses (wells 9A) are formed on the surface of a low cell-adhesion substrate (plate 8). The bottom surfaces 92 of the plurality of wells 9A, which are furthest from the surface (top surface 90) of plate 8, are flat surfaces that extend along the top surface 90. As with the above, this also improves the positional accuracy of the droplets and cells when forming spheroids in the wells 9A, and enhances the self-aggregation of the cells C in the first coating liquid A.

[0081] In the above-described method for controlling cell arrangement, a plurality of recesses (wells 9A) are formed on the surface of the low cell-adhesion substrate (plate 8). The bottom surfaces 92 of the plurality of wells 9A, which are furthest from the surface (top surface 90) of the plate 8, are curved. That is, the bottom surfaces 92 may have a U-shaped cross section. As with the above, this also improves the positional accuracy of the droplets and cells during spheroid formation, and enhances the self-aggregation of the cells C in the first coating liquid A. Note that since the bottom surface 92 is curved (e.g., a part of a sphere), the self-aggregation of the cells C is further enhanced compared to when it is flat.

[0082] In the above-described method for controlling cell arrangement, a plurality of recesses (wells 9A) are formed on the surface of a low cell-adhesion substrate (plate 8). The bottom surfaces 92 of the plurality of wells 9A, which are furthest from the surface (top surface 90) of the plate 8, have walls that are inclined relative to the direction perpendicular to the top surface 90 (the up-down direction). The portion of the wall furthest from the top surface 90 has a pointed shape. In other words, the bottom surface 92 may have a V-shaped cross section. As described above, this also improves the positional accuracy of the droplets and cells during spheroid formation and enhances the self-aggregation of the cells C in the first coating liquid A. Note that, since the bottom surface 92 is curved (e.g., part of the side surface of a cone), the self-aggregation of the cells C is further enhanced compared to when it is flat (e.g., part of the side surface of a pyramid).

[0083] In the above-described method for controlling cell arrangement, a plurality of recesses (depressions 9B) are formed on the surface (top surface 90 of the bottom surface) of the low cell-adhesion substrate (plate 8). The maximum dimension of the recesses 9B in a planar view is less than 1 mm. As with the above, this also improves the positional accuracy of the droplets and cells during spheroid formation, and enhances the self-aggregation of the cells C in the first coating liquid A. Furthermore, by having the recesses 9B on the top surface 90 of the bottom surface of the dish, the self-aggregation of the cells C can be further enhanced compared to when the top surface 90 is flat and does not have any recesses.

[0084] As a modified example, a depression 9B as shown in Figure 8 may be formed on the bottom surface 92 of the well 9A in Figures 4 to 6. This further enhances the self-aggregation of the cells C with each other. [Example]

[0085] An experiment was conducted to actually form spheroids based on the cell arrangement control method of this embodiment. In this experiment, coating solutions were prepared as follows using an apparatus capable of supplying multiple coating solutions, such as the coating apparatus 100 shown in Figure 1.

[0086] The plate 8 used was a multi-well plate having 96 wells 9A as shown in Figure 3. Each droplet described below was supplied into the wells 9A.

[0087] The first coating solution A used RPMI-1640 as the first solvent and contained 0.7 mg / mL collagen type I-A as the gel raw material. 8 The second coating solution B contained phosphate-buffered saline (+) (PBS(+)) as the second solvent. This solvent contained methylcellulose as a viscosity enhancer. The medium M was FGM-3.

[0088] Using a 1000 μm diameter needle of the needle coating mechanism (see needle coating mechanism 104 in Figure 1 and coating needle 24 in Figure 2), the first coating liquid A was applied to one well 9A of the plate 8. Next, the second coating liquid B was dripped onto the first coating liquid A to cover it. The second coating liquid B was dripped onto the wells using a dispenser (see drip mechanism 105 in Figure 1). Then, medium M was dripped onto the wells to cover them. Medium M was dripped onto the wells using a manual pipette. Spheroids were formed by culturing the cells in medium M. This process is as described in the section (Explanation of the specific manufacturing method using diagrams).

[0089] Figure 20 shows phase contrast microscope images of the coating solution in Example 1 immediately after application and after culture. As shown in Figure 20, the diameter of the coating solution is smaller after culture (Day 6) compared to immediately after application (Day 0). As shown in Figure 20, a large number of cells C in the coating solution A self-aggregated during culture, resulting in spheroids. Note that when the coating solution (cell tissue) is non-spherical and close to spherical, the above diameter is calculated as the diameter assuming that it is a sphere of the same volume. [Example]

[0090] An experiment was conducted to form spheroids by applying multiple doses of the first coating liquid A. The types of plate 8, first coating liquid A, second coating liquid B, and medium M used in this experiment were the same as in Example 1. However, in this experiment, the first coating liquid A was divided into two types. The two types of first coating liquid were first coating liquid A1 and first coating liquid A2 (see Figure 10). Using a cell tracker, cells contained in the first coating liquid A1 were stained red, and cells contained in the first coating liquid A2 were stained green.

[0091] Using a 1000 μm diameter coating needle of a needle coating mechanism (see needle coating mechanism 104 in FIG. 1 and coating needle 24 in FIG. 2), a first coating liquid A1 was coated into one well 9A of a plate 8. Next, a first coating liquid A2 was coated adjacent to the first coating liquid A1 in the same well 9A to which the first coating liquid A1 was coated. FIG. 21 is a schematic diagram showing the coating manner of the first coating liquid A1 and the first coating liquid A2 in Example 2. As shown in FIG. 21(A), one sample each of the first coating liquid A1 and the first coating liquid A2 was coated, and as shown in FIG. 21(B), two samples each of the first coating liquid A1 and the first coating liquid A2 were coated. In FIG. 21(A), one sample each of the first coating liquid A1 and the first coating liquid A2 is arranged so that they come into contact with each other. In FIG. 21(B), two samples of the first coating liquid A1 are arranged so that they come into contact with each other. The two first coating liquids A2 are arranged so as to sandwich the two first coating liquids A1. Both of the two first coating liquids A2 are in contact with the first coating liquid A1. One first coating liquid A2 and the other first coating liquid A2 are not in contact with each other. As a result, the two first coating liquids A1 and the two first coating liquids A2 are arranged at the four corners of a quadrangle, with the two first coating liquids A1 on one diagonal and the two first coating liquids A2 on the other diagonal. Thereafter, the second coating liquid B and the medium M were dropped onto both of these, as in Example 1, and cells C were cultured to form spheroids.

[0092] Figure 22 shows phase-contrast and fluorescent microscope images immediately after application of the coating liquid and after culturing in Example 2. Figure 22(A) corresponds to an example in which two types of coating liquids were applied, one each, as in Figure 21(A). Figure 22(B) corresponds to an example in which two types of coating liquids were applied, two each, as in Figure 21(B). In both Figures 22(A) and 22(B), the left side is a phase-contrast microscope image, and the right side is a fluorescent microscope image. As shown in Figure 22(A), two spheroids were applied, one each of the red first coating liquid A1 and the green first coating liquid A2. This resulted in spheroids in which red-stained cellular tissue and green-stained cellular tissue were localized. As shown in Figure 22(B), four spheroids were applied, two each of the red first coating liquid A1 and the green first coating liquid A2. Two red-stained cellular tissues were connected and appeared as one. Two green-stained cellular tissues were localized, flanking the red cellular tissue. These cellular tissues were connected to form a unified spheroid.

[0093] As can be seen from (A) and (B) of Figure 22, the method of this embodiment enabled the formation of spheroids in which cells self-aggregated at high density with high positional accuracy, regardless of whether the number of first application liquid A was two or three or more (four). [Example]

[0094] An experiment similar to that of Example 2 was conducted. The type of plate 8 and second coating liquid B used in this experiment were the same as those used in Examples 1 and 2. However, the first coating liquid A used in this experiment differed from those used in Examples 1 and 2 in that it contained normal human dermal fibroblasts. The cell concentration and each material in the first coating liquid A other than the cells were the same as those used in Examples 1 and 2. Furthermore, in this experiment, medium M was prepared by adding FBS to DMEM, with FBS accounting for 10% by volume (10 v / v%) of the total medium M. In other words, medium M prepared by adding 10 ml of FBS to 90 ml of DMEM was used. Only in Example 3 was a coating needle with a diameter of 500 μm used.

[0095] The supply method for each coating liquid was the same as in Example 2. FIG. 23 is a schematic diagram showing the application of the first coating liquid A1 and the first coating liquid A2 in Example 3. As shown in FIG. 23(A), a coating was prepared in which a total of three coating liquids, two of the first coating liquid A1 and one of the first coating liquid A2, were arranged in a straight line. As shown in FIG. 23(A), two of the first coating liquid A1 sandwiched one of the first coating liquid A2, forming a straight line, and the three coating liquids were in contact with each other. Also, as shown in FIG. 23(B), a coating was prepared in which a total of four coating liquids, two of the first coating liquid A1 and two of the first coating liquid A2, were arranged in a straight line. As shown in FIG. 23(B), the first coating liquid A1 and the first coating liquid A2 were alternately arranged to form a straight line, and the four coating liquids were in contact with each other.

[0096] Figure 24 shows phase-contrast and fluorescence microscope images immediately after application of the coating solution and after culture in Example 3. Figure 24 (A) corresponds to an example in which three coating solutions were supplied in a straight line, as in Figure 23 (A). Figure 24 (B) corresponds to an example in which four coating solutions were supplied in a straight line, as in Figure 23 (B). In both Figures 24 (A) and (B), only the bottom row of the three rows is a fluorescence microscope image, and the others are phase-contrast microscope images. In Figure 24, the image immediately after application is indicated as "0 h," and the image after culture is indicated as "4 h." As shown in Figures 24 (A) and (B), spheroids with arrangements corresponding to Figures 23 (A) and (B) were obtained.

[0097] The features described in the above-described embodiments (each example) may be applied in appropriate combinations within the scope of technical compatibility.

[0098] The embodiments disclosed herein should be considered to be illustrative in all respects and not restrictive. The scope of the present invention is defined by the claims, not by the above description, and is intended to include all modifications within the meaning and scope of the claims.

[0099] (Addendum) Various aspects of the present disclosure are summarized below as appendices.

[0100] (Appendix 1) providing a substrate for providing cells to be cultured; a first coating liquid application step of applying a first coating liquid containing a first solvent onto the substrate at least once; a second coating liquid dropping step of dropping a second coating liquid containing a thickener and a second solvent so as to cover the first coating liquid; the cells are included in at least one application of the first coating liquid in the first coating liquid application step; The method for controlling cell arrangement, wherein the substrate is a low cell adhesion substrate.

[0101] (Appendix 2) The method for controlling cell arrangement described in Appendix 1, wherein the low cell-adhesion substrate is formed by coating the surface of the substrate with either MPC polymer or P-HEMA.

[0102] (Appendix 3) The method for controlling cell arrangement described in Appendix 1, further comprising a step of controlling the contact angle of the surface with water by irradiating the surface of the substrate with radiation.

[0103] (Appendix 4) a plurality of recesses are formed on the surface of the low cell-adhesion substrate; The method for controlling cell arrangement according to any one of appendix 1 to 3, wherein the bottom surfaces of the plurality of wells farthest from the surface are flat surfaces extending along the surface.

[0104] (Appendix 5) a plurality of recesses are formed on the surface of the low cell-adhesion substrate; 4. The method for controlling cell arrangement according to any one of claims 1 to 3, wherein the bottom surfaces of the plurality of wells farthest from the surface are curved.

[0105] (Appendix 6) a plurality of recesses are formed on the surface of the low cell-adhesion substrate; A method for controlling cell placement described in any one of Appendix 1 to 3, wherein the bottom surfaces of the plurality of recesses farthest from the surface are wall surfaces that are inclined with respect to a direction perpendicular to the surface, and the portion of the wall surface farthest from the surface has a pointed shape.

[0106] (Appendix 7) a plurality of recesses are formed on the surface of the low cell-adhesion substrate; 4. The method for controlling cell arrangement according to any one of claims 1 to 3, wherein the maximum dimension of the recess in plan view is less than 1 mm.

[0107] (Appendix 8) The method for controlling cell arrangement according to any one of appendices 1 to 7, wherein in the step of applying the first application liquid onto the substrate, the first application liquid is applied onto the substrate three or more times and the cells are arranged on the substrate.

[0108] (Appendix 9) In the step of coating the substrate, the first coating liquid is coated in N amounts (N is a natural number of 2 or more), In the step of coating the substrate, the dimensions of two first coating liquids arbitrarily selected from the N first coating liquids are defined as r 1n , r 1(n+1) (n is a natural number in the range of 1≦n≦N−1), and the distance between the centroids of the two first liquids is d n,n+1 given that, (Number 4)d n,n+1 ≦r 1(n+1) -r 1n ···(1) and (number 5)r 1(n+1) -r 1n <d n,n+1 <r 1n +r 1(n+1) ···(2) The method for controlling cell arrangement according to any one of appendices 1 to 8, wherein the first application liquid is applied to a position where any one of the following conditions is satisfied.

[0109] (Appendix 10) 10. The method for controlling cell arrangement according to claim 9, wherein when N pieces of the first coating liquid are coated, the components of the N pieces of the first coating liquid are identical to each other.

[0110] (Appendix 11) 10. The method for controlling cell arrangement according to claim 9, wherein when N pieces of the first coating liquid are coated, the components of the N pieces of the first coating liquid are different from each other.

[0111] (Appendix 12) The method for controlling cell arrangement according to any one of appendices 9 to 11, wherein, when N pieces of the first coating liquid are coated, the types of cells contained in at least two of the N pieces of the first coating liquid are different from each other.

[0112] (Appendix 13) The method for controlling cell placement according to any one of appendices 1 to 12, wherein the step of applying the solution onto the substrate uses any one selected from the group consisting of a needle-type bioprinter, a dispenser, an inkjet, and a pipette. [Explanation of symbols]

[0113] 8 plate, 8A plate body, 9A well, 9B recess, 20 coating needle holder, 21 coating material container, 35 movable base, 41 servo meter, 43 cam, 44 bearing, 45 cam connecting plate, 46 movable part, 90 top surface, 91 wall surface, 92 bottom surface, 93 curved surface, 94 inclined wall surface, 100 coating device, 101 X-axis stage, 102 Y-axis stage, 104, 104-1, 104-2 needle coating mechanism, 105, 105-1, 105-2 drip mechanism, 106 observation optical system, 107 coating mechanism, A, A1, A2 first coating liquid, B second coating liquid, C cell, C1 cardiomyocyte, C2 cardiac fibroblast.

Claims

1. providing a substrate for providing cells to be cultured; a first coating liquid coating step of coating the substrate at least once with a first coating liquid containing a first solvent; a second coating liquid dropping step of dropping a second coating liquid containing a thickener and a second solvent onto the first coating liquid so as to cover the first coating liquid; the cells are contained in at least one application of the first coating liquid in the first coating liquid application step; The method for controlling cell arrangement, wherein the substrate is a low cell adhesion substrate.

2. 2. The method for controlling cell arrangement according to claim 1, wherein the low cell-adhesion substrate is formed by coating the surface of the substrate with either MPC polymer or P-HEMA.

3. The method for controlling cell arrangement according to claim 1 , further comprising the step of controlling the contact angle of the surface with water by irradiating the surface of the substrate with radiation.

4. a plurality of recesses are formed on the surface of the low cell-adhesion substrate; The method for controlling cell arrangement according to claim 1 or 2, wherein the bottom surfaces of the plurality of wells farthest from the surface are flat surfaces extending along the surface.

5. a plurality of recesses are formed on the surface of the low cell-adhesion substrate; The method for controlling cell arrangement according to claim 1 or 2, wherein the bottom surfaces of the plurality of wells farthest from the surface are curved.

6. a plurality of recesses are formed on the surface of the low cell-adhesion substrate; The method for controlling cell placement described in claim 1 or 2, wherein the bottom surfaces of the plurality of recesses farthest from the surface are wall surfaces inclined with respect to a direction perpendicular to the surface, and the portion of the wall surface farthest from the surface has a pointed shape.

7. a plurality of recesses are formed on the surface of the low cell-adhesion substrate; The method for controlling cell arrangement according to claim 1 or 2, wherein the maximum dimension of the recess in plan view is less than 1 mm.

8. The method for controlling cell arrangement according to claim 1 or 2, wherein in the step of applying the first application liquid onto the substrate, the first application liquid is applied onto the substrate three or more times and the cells are arranged on the substrate.

9. In the step of coating the substrate, the first coating liquid is coated in N amounts (N is a natural number of 2 or more), In the step of coating the substrate, the dimensions of two of the first coating liquids arbitrarily selected from the N first coating liquids are defined as r １ｎ , r １（ｎ＋１） (n is a natural number satisfying the condition 1≦n≦N−1), and the distance between the centroids of the two first coating liquids is d ｎ，ｎ＋１ given that, (Number 1) d ｎ，ｎ＋１ ≦r １（ｎ＋１） -r １ｎ ・・・(1) and (number 2) r １（ｎ＋１） -r １ｎ <d ｎ，ｎ＋１ <r １ｎ +r １（ｎ＋１） ・・・(2) The method for controlling cell arrangement according to claim 1 or 2, wherein the first application liquid is applied to a position where any one of the following conditions is satisfied:

10. The method for controlling cell arrangement according to claim 9 , wherein when N first coating liquids are coated, the constituent components of the N first coating liquids are identical to each other.

11. The method for controlling cell arrangement according to claim 9 , wherein when N first coating liquids are coated, the constituent components of the N first coating liquids are different from each other.

12. The method for controlling cell arrangement according to claim 9 , wherein when N pieces of the first coating liquid are coated, the types of the cells contained in at least two of the N pieces of the first coating liquid are different from each other.

13. The method for controlling cell placement according to claim 1 or 2, wherein the step of applying the solution onto the substrate uses any one selected from the group consisting of a needle-type bioprinter, a dispenser, an inkjet, and a pipette.

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