Method for producing cell tissue

Abstract

The depth, relative to a surface, of an application surface of a first substrate for cell tissue formation is measured. Prior to the supply of a cell tissue to the first substrate, at least one application section, which is a section of the application surface of the first substrate, is subjected to first trial application (S3) with a first application liquid. In the step (S3) for the first trial application, the first application liquid is applied (S3-1) to the one application section using a first pressing amount of an application needle, said first pressing amount corresponding to the depth. A first measurement step (S3-3) for measuring the diameter of the first application liquid is performed.

Description

Method for producing cell tissue

[0001] The present invention relates to a method for producing a cell tissue.

[0002] Japanese Patent No. 6461260 (Patent Document 1) discloses a method for storing the focus position of the substrate, which is the object to be coated, during the process of creating a circuit pattern using a coating needle type coating device, and correcting the vertical position of the coating mechanism.

[0003] Patent No. 6461260

[0004] Multiwell plates with multiple wells are used to create (manufacture) cell tissues. The height of the bottoms of the multiple wells (well bottoms) in commercially available multiwell plates is not uniform and varies. For example, the height of the well bottoms of a 96-well plate can vary by as much as 150 μm within a single plate. When creating cell tissues using a coating mechanism in a series of operations in all wells of a plate with different bottom heights, setting the focus under the assumption that the bottom positions of all wells are the same results in variations in the amount of coating liquid applied to each well. Here, the coating amount refers to the amount of coating liquid transferred from the coating needle to the well. This is because the difference in the height of the bottoms of each well causes the amount of pressure the lowered coating mechanism exerts on the well bottoms to vary between individual wells.

[0005] Furthermore, even if the height of the well bottoms is the same, variations in the amount of coating liquid attached to the coating needle can cause variations in the amount of coating liquid applied to each well. Patent Document 1 simply stores the focus position of the substrate in advance. Patent Document 1 does not take into account the variations in the height of the well bottoms and variations in the amount of coating liquid attached to the coating needle.

[0006] 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 producing cell tissue that can reduce variations in the amount of application among a plurality of application sections.

[0007] In a method for producing cellular tissue according to the present disclosure, a first substrate for forming cellular tissue is placed in equipment. The depth of the coating surface of the first substrate relative to the surface of the first substrate is measured. Prior to supplying the cellular tissue to the first substrate, a first trial coating step is performed with a first coating liquid on at least one coating portion that is a part of the coating surface of the first substrate. In the first trial coating step, the first coating liquid is applied to one coating portion with a first depression amount of the coating needle corresponding to the depth. A first measurement step is performed to measure the diameter of the first coating liquid.

[0008] According to the present disclosure, a first trial application is performed based on the measurement results of the depth of the application surface prior to supplying the cellular tissue to the first substrate, thereby providing a method for producing cellular tissue that can reduce variation in the amount of application among multiple application sections.

[0009] 1A-1).

[0023] FIG. 1B is a schematic front view of a coating device according to a first embodiment.

[0024] FIG. 1C is a schematic diagram showing a needle coating mechanism of the coating device shown in FIG. 1.

[0025] FIG. 1D is a schematic perspective view of a plate.

[0026] FIG. 1E is a schematic cross-sectional view showing a first example of the shape of wells formed in the plate.

[0027] FIG. 1F is a schematic cross-sectional view showing a second example of the shape of wells formed in the plate.

[0028] FIG. 1G is a flowchart showing an outline of a method for producing cell tissue according to the present embodiment.

[0029] FIG. 1H is a flowchart of a step (S1) performed on a first substrate.

[0030] FIG. 1I is a schematic diagram for explaining alignment correction in step (S1A-1).

[0031] FIG. 1I is a flowchart of a step (S1) performed on a second substrate.

[0032] FIG. 1I is a flowchart of a mounting confirmation step (S2).

[0033] FIG. 1I is a flowchart of trial coating of a first coating liquid (S3).

[0034] FIG. 1J is a schematic diagram showing the XY coordinates of the trial-coated first coating liquid and the XY coordinates of its target position.

[0035] FIG. 1J is a schematic diagram for explaining a first determination step (S3-4) showing the relationship between an area to which the first coating liquid should be trial-coated and an area to which the first coating liquid is actually applied.

[0036] FIG. 1J is a flowchart of trial supply (S4) of a second coating liquid.

[0037] FIG. 1J is a schematic diagram showing an example of selection of wells, of which bottom depths are to be measured, from wells included in a multiwell plate serving as a first substrate. 28 is a schematic diagram for explaining a method for estimating the depth of the bottom of a well whose depth has not been measured. FIG. 29 is a schematic diagram showing the arrangement of 96 wells included in a plate. FIG. 29 is a graph showing the measured values ​​of the bottom height for each well of the plate of FIG. 17. FIG. 29 is a schematic diagram showing the variation in the amount of adhesion of a first coating liquid to a coating needle. FIG. 29 is a schematic diagram showing the state before the first coating liquid is applied in an embodiment. FIG. 30 is a schematic diagram showing the composition of the first coating liquid in an embodiment. FIG. 31 is a schematic diagram showing the process of applying the first coating liquid in an embodiment. FIG. 32 is a schematic diagram showing the state after the first coating liquid has been applied in an embodiment. FIG. 33 is a schematic diagram showing the process of supplying a second coating liquid in an embodiment. FIG. 34 is a schematic diagram showing the state inside a well after the process of FIG. 24 has been performed. FIG. 35 is a schematic diagram showing the process of supplying a culture medium in an embodiment. FIG. 36 is a schematic diagram showing the state of cells in the first coating liquid before culture. FIG. 37 is a schematic diagram showing the state of cells in the first coating liquid after culture. FIG. 38 is a phase contrast microscope image immediately after application of the coating liquid in Example 1.

[0010] (Introduction) First, a brief description of the method for producing cellular tissue according to this embodiment will be given. As shown in FIG. 3 , a first substrate for forming cellular tissue, e.g., a plate 8 having a plurality of wells 9A, is placed in the equipment. As shown in FIGS. 4 and 5 , the depth of the application surface (bottom surface 92) of at least one of the plurality of wells 9A in the plate 8 relative to the surface 8B of the plate 8 is measured. As shown in FIG. 11 , prior to supplying cellular tissue to the plate 8, a first trial application of a first application liquid A is performed on an application portion, which is a portion of at least one application surface (bottom surface 92) of the plate 8. In the first trial application step, the first application liquid A is applied to a portion (application portion) of the bottom surface 92 of one well 9A with a first depression amount of the application needle 24 corresponding to the depth. The diameter of the first application liquid A is measured (first measurement step).

[0011] The method for producing cell tissue may further include the following: calculating a first difference between the diameter obtained in the first measurement step and a target diameter, which is a target value of the diameter, and determining whether or not the first depression amount needs to be changed based on the first difference (first determination step). If it is determined in the first determination that the first depression amount needs to be changed, a second trial application step of applying the first application liquid to the application portion of one well 9A by a second depression amount different from the first depression amount is further performed after the first trial application step.

[0012] (Embodiment 1) (Configuration of Coating Apparatus) FIG. 1 is a schematic front view showing a coating apparatus according to embodiment 1. For convenience of explanation, the X, Y, and Z directions are introduced. Referring to FIG. 1, 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. As described above, in this specification, "coating" includes both supplying the coating material using a coating needle, as described below, and supplying the coating material by dripping. For this reason, the former supply using a coating needle may be referred to as "needle coating" in this specification. Coating apparatus 100 in FIG. 1 includes one needle coating mechanism 104 and one dripping mechanism 105. The needle coating mechanism 104 and the dripping mechanism 105 are arranged at a distance in the X direction. The distance between them in the X direction is constant and does not change.

[0013] The trial coating, which is the subject of this embodiment, is performed on the first and second coating liquids, which are bioinks, but not on the culture medium, and therefore the dripping mechanism for the culture medium is not shown in Figure 1.

[0014] The X-axis stage 101 (stage) 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.

[0015] 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 and other objects using visible light. However, the observation of the plate 8 and other objects is not limited to visible light; infrared light, X-rays, ultrasound, and other methods may also be used. Depending on the material of the plate 8, the plate 8 may also be observed using magnetism. The plate 8 observed by means other than visible light does not need to be transparent or translucent and may be opaque.

[0016] Fig. 2 is a schematic diagram showing the needle coating mechanism of the coating device shown in Fig. 1. Referring to 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 that holds the coating needle holder 20, and a coating material container 21. 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.

[0017] In the needle coating mechanism 104, the servo motor 41 is installed with its central axis extending along 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 connected to the rotation shaft of the servo motor 41 and a flange connected to one end of the center. The upper surface of the flange (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 and slopes so that the distance from the bottom surface of the flange varies. Specifically, the cam surface includes an upper flat region (thickest) that is farthest from the bottom surface, a lower flat region spaced apart from the upper flat region, and a sloped portion that smoothly connects the upper flat region and the lower flat region. The lower flat region is the region closest to the bottom surface (thinnest).

[0018] A bearing 44 is disposed so as to contact the cam surface of the cam 43. A cam connecting plate 45 is connected to the 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 the movable part 46. A coating needle holder 20 is installed on the 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 disposed on the underside of the coating needle holder 20 (the lower side opposite the side where the servo motor 41 is located) so as to protrude from the coating needle holder 20. A coating material container 21 is disposed below the coating needle holder 20. The coating needle 24 is held in an inserted state in the coating material container 21.

[0019] 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.

[0020] Furthermore, the movable portion 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.

[0021] 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 in the Z-axis direction, which is in contact with the cam surface of the cam 43, fluctuates 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 fluctuation 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 located in the upper position 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 is attached to the tip of the application needle 24. The liquid material thus attached is transferred to the inside of the well 9A of the plate 8, thereby achieving a highly reproducible application process.

[0022] Fig. 3 is a schematic perspective view of a plate. Referring to Fig. 3, in this embodiment, a coating material as a liquid material is coated and supplied into the interior of a plurality of wells 9A formed in plate 8. However, the object to which the coating material is supplied is not limited to this. Plate 8 has a thickness in the Z-axis direction, and a plurality of wells 9A are formed on its uppermost surface. The plurality of wells 9A are recessed portions of the upper surface of plate 8. The plurality of wells 9A may be formed at intervals from one another, for example, in eight rows in the X direction and twelve columns in the Y direction in Fig. 3, for a total of 96 wells. The planar shape of wells 9A may be any shape, such as a circle.

[0023] 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, multiple 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 with surface 8B and bottom surfaces 92 extending along surface 8B. Bottom surfaces 92 are the flat surfaces of wells 9A located at the farthest position from surface 8B. Wells 9A are formed by bottom surfaces 92 and wall surfaces 91 that are continuous with the outer edge of bottom surfaces 92 and extend to intersect with bottom surfaces 92.

[0024] 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 8B. Therefore, the area of ​​the bottom surface 92 is smaller than the area of ​​the opening in the surface 8B 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 surface 8B. That is, in the cross section of FIG. 4 , the wall surface 91 may be perpendicular to the surface 8B, or may be inclined and not perpendicular to the surface 8B. 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 FIG. 5 . 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.

[0025] 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 connect to the lowest part of the wall surface 91. The well 9A in FIG. 5 has a curved bottom surface 92 (the bottom part farthest from the surface 8B). 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.

[0026] When the well 9A of Fig. 5 is used, the height of the bottom surface 92 may be calculated, for example, as the vertical coordinate of the lowest point P of the bottom surface 92. Alternatively, it may be calculated as the average value of the vertical coordinate of the area defined as the bottom surface 92 (the area below the bent position relative to the wall surface 91). However, from the perspective of simplifying the explanation below, it is assumed that the bottom surface 92 of the well 9A is flat, as shown in Fig. 4, and that the height position (depth) of the bottom surface 92 is constant throughout. In other words, as shown in Fig. 4, the bottom surface 92 of the well 9A is assumed to extend horizontally so as to be approximately parallel to the surface 8B.

[0027] (Method for Producing Cellular Tissue) FIG. 6 is a flowchart outlining the method for producing cellular tissue according to this embodiment. As shown in FIG. 6, a substrate is first placed in the equipment (S0). The substrate placed in the equipment here is the same as the substrate on which the actual coating will be performed later, such as plate 8 (a multiwell plate having multiple wells) as shown in FIG. 3. Here, "equipment" refers to coating device 100. The actual coating refers to the actual coating, not the trial coating, that is, the coating performed to actually produce cellular tissue. The substrate on which the actual coating will be performed later, in other words, the substrate for forming cellular tissue, will be referred to as the "first substrate" hereinafter. The first substrate is always prepared in step (S0). That is, the first substrate is placed in the equipment in step (S0). As described below, the first substrate is not limited to a multiwell plate and may be a flat glass slide or the like. However, for simplicity of explanation, this embodiment will be described assuming that the first substrate is a multiwell plate and that the coating will be performed on the bottom surfaces of the wells.

[0028] In step (S0), a second substrate may be prepared in addition to the first substrate. That is, in step (S0), a second substrate may be placed in the equipment in addition to the first substrate. The second substrate is not used for actual application, but is used only for trial application. That is, the second substrate is not used for cell tissue formation. The second substrate may be the same type of plate 8 (multiwell plate) as the first substrate. However, the second substrate is not limited to this. The second substrate may be any one selected from the group consisting of dishes of various diameters, microchannels, slide glasses, cell desks LF, and PDMS substrates. When the second substrate is not placed in the equipment, the first substrate is used for both trial application and actual application. In this embodiment, for simplicity of explanation, the second substrate is assumed to be a plate without wells.

[0029] Next, the depth of the coating surface is measured (S1). That is, in step (S0), the depth of the coating surface of the first substrate placed in the equipment relative to the surface of the first substrate is measured. The coating surface is the surface of the first substrate to which the coating liquid is transferred and supplied. The coating surface is, for example, the bottom surface 92 of at least one of the multiple wells 9A formed in the plate 8. Here, the first meaning of "depth" refers to the position coordinate (height) of the bottom surface 92 (coating surface) of the well 9A in the vertical direction (Z direction). In other words, the "depth" of the bottom surface of the well 9A is the vertical coordinate of the bottom surface 92 (coating surface). For example, the "depth" may be the Z-direction coordinate (negative value) of the bottom surface 92 when the Z-direction coordinate of the surface of the first substrate (surface 8B in FIG. 4) is set to a reference value of zero. If the first substrate does not have a well 9A, the "depth" may also be considered the Z-direction coordinate of the coating portion (described below) relative to the surface of the first substrate (a portion other than the coating portion (described below)). Alternatively, the second meaning of "depth" may be the amount of step (a difference in height that is a positive value) between the surface and the bottom surface caused by the portion of well 9A being recessed relative to the surface of the first substrate. In the rest of this specification, the term "depth" will be used mainly in the first meaning described above.

[0030] 4, the application surface is the bottom surface 92 of the well 9A. If the first substrate is a flat plate without wells 9A, the application surface is its main surface.

[0031] 7 is a flowchart of step (S1) performed on the first substrate. As shown in FIG. 7, step (S1) performed on the first substrate will be referred to as step (S1A) hereinafter. In step (S1A), when trial coating is performed on the first substrate, the vertical position (height: depth) of the coating surface is measured in advance. Step (S1A) for the first substrate may be referred to as (a) hereinafter. In step (S1A) for the first substrate, the position (depth) in the height direction (Z direction) of the well bottom surface (coating surface) to which the first coating liquid and the second coating liquid are to be trial coated in the subsequent trial coating step is measured.

[0032] In step (S1A), alignment correction of the first substrate is first performed (S1A-1). Specifically, XY coordinate data of an arbitrary position on plate 8 (multiwell plate) in FIG. 3 is first set in advance. XY coordinates refer to X and Y coordinates (coordinate positions in a plan view). Next, plate 8 is placed on XY stage 101. The XY coordinates of the position of the actually placed plate 8 are measured.

[0033] FIG. 8 is a schematic diagram illustrating alignment correction in step (S1A-1). FIG. 8 shows a plan view of plate 8 viewed from a direction substantially perpendicular to the XY plane. As shown in FIG. 8, plate 8 is pre-configured in coating apparatus 100 to be positioned at set position 81. Any portion of plate 8 at set position 81 (e.g., any one well) is configured with its outline located at set well position 9A1. As an example, FIG. 8 shows set well positions 9A1 for a pair of diagonally opposing corner wells. However, based on measurements in this step, plate 8 is actually positioned at actual measurement position 82. Based on these measurements, well 9A corresponding to set well position 9A1 is positioned at actual measurement well position 9A2. In this case, the error between set position 81 and actual measurement position 82, and the error between set well position 9A1 and actual measurement well position 9A2, are corrected as misalignment. This correction of misalignment is referred to as alignment correction.

[0034] Referring again to FIG. 7 , after alignment correction, the height position of the coating surface, such as the well bottom height, is measured (S1A-2). That is, the vertical (Z-direction) height coordinate of the bottom surface 92 (see FIG. 4) of the well 9A (see FIG. 3) included in the multiwell plate, which is the first substrate, is measured. This measurement may be performed using a laser sensor. Alternatively, this measurement may be performed using the autofocus of a camera. Using these methods, the Z-direction position can be measured with high precision. The measured height position of the coating surface (such as the well bottom) is stored as the depth of the bottom surface 92 relative to the surface 8B (S1A-3). Alternatively, the height of the well bottom may be measured by acquiring an image using a combination of illumination and an observation device such as a camera, and then processing the image.

[0035] FIG. 9 is a flowchart of step (S1) performed on the second substrate. Step (S1) is not limited to being performed on the first substrate. In other words, as shown in FIG. 9, step (S1) may also be performed on the second substrate. Step (S1) performed on the second substrate will be referred to as step (S1B) below. When trial coating is performed on the second substrate, step (S1B) is a step in which the vertical position (height: depth) of the coating surface is measured in advance. Step (S1B) on the second substrate will sometimes be referred to as (a)' below.

[0036] In step (S1B), alignment correction of the second substrate is first performed (S1B-1), especially when the second substrate is a multi-well plate and trial coating is to be performed within the wells. However, if the second substrate does not have wells, such as a glass slide that is a flat surface of uniform height, alignment correction of the second substrate is not necessary. With a glass slide or the like, the Z coordinate of the coating surface is approximately the same throughout, regardless of the position on the XY plane where trial coating is performed. Therefore, information on the position on the XY plane is not necessary.

[0037] The height position of the coated surface is measured for the second substrate (S1B-2). The procedure is the same as in step (S1A-2). In step (S1B-2), the position (depth) in the height direction (Z direction) of only a portion (coated portion) of the coated surface of the well bottom or slide glass, etc., is measured.

[0038] No actual coating is performed on the second substrate. Therefore, the process of estimating the position of the well bottom, which will be described later, is not performed on the second substrate. Storing the measured Z-direction positions of the well bottoms is necessary for estimating the Z-direction positions of the other well bottoms that have not been measured, as will be described in the following second embodiment. Therefore, there is no need to store the measured Z-direction positions of the well bottoms for the second substrate, for which no actual coating is performed and for which only the relationship between the Z-direction height and the coating diameter needs to be known.

[0039] Referring again to FIG. 6, the installation confirmation (S2) is performed in the coating mechanism. FIG. 10 is a flowchart of the installation confirmation step (S2). As shown in FIG. 10, the needle coating mechanism 104 is installed in the coating device 100. The coating needle 24 and the coating material container 21 are installed in the needle coating mechanism 104 (S2-1). It is confirmed whether the coating needle 24 installed in the needle coating mechanism 104 moves normally in the vertical direction (Z direction) (S2-2). If there is a problem with the vertical movement of the coating needle 24, the process proceeds from step (S2-2) in the direction of NOT GOOD, as indicated by the arrow. In other words, the process returns from step (S2-2) to step (S2-1). The coating needle 24 is re-installed, and the drive confirmation (S2-2) is performed again. Steps (S2-1) and (S2-2) are repeated until the drive status of the coating needle 24 is determined to be good.

[0040] If there is no problem with the up-and-down movement of the coating needle 24, the process proceeds from step (S2-2) in the direction of GOOD, as indicated by the arrow. That is, the drip mechanism 105 is attached to the coating device 100 (S2-3). The ejection time and ejection pressure of the second coating liquid from the drip mechanism 105 are set (S2-4). The order of steps (S2-1) and (S2-2) and steps (S2-3) and (S2-4) may be reversed. Step (S2) may hereinafter be referred to as (b).

[0041] 6 again, trial application of the first coating liquid is performed (S3). This is a step in which, prior to the actual application for supplying the cell tissue to the first substrate, the first coating liquid is trial-applied (trially applied) from the needle application mechanism 104 to a coating portion, which is a part of the bottom surface 92 of at least one well 9A of the first substrate.

[0042] The first coating liquid may be trial-coated to the well 9A whose bottom surface 92 has its depth measured in step (S1), or to a different well 9A. Alternatively, the first coating liquid may be trial-coated to a second substrate whose bottom surface 92 has its depth measured in step (S1) (different from the first substrate to be actually coated). In this case, the first coating liquid is necessarily trial-coated to a position different from the well 9A whose depth has been measured. In either case, the first coating liquid is trial-coated to a location whose Z-direction position on the coating surface (such as the bottom surface of a well or the surface of a slide glass) has been measured in step (S1).

[0043] When the first substrate is used, the first coating liquid is applied to the coating portion of any well 9A whose Z direction position has been measured, other than the well 9A to be used for the main coating, which is formed on the plate 8. When the second substrate is used, the first coating liquid is applied to the position (coating portion) whose Z direction position has been measured.

[0044] 11 is a flowchart of the trial application of the first coating liquid (S3). As shown in FIG. 11, in the trial application step (S3) of the first coating liquid, the first coating liquid is first applied by a first pushing amount (S3-1). The first pushing amount refers to the amount (distance) by which the coating needle 24 is lowered in the vertical direction (Z direction) to apply the first coating liquid to the coating surface. The amount by which the coating needle 24 is lowered is an amount that depends on the position of the coating surface in the Z direction, such as the depth of the bottom of the well to which the first coating liquid is applied.

[0045] The first coating liquid preferably contains cells, a gel raw material, and a solvent as its main components, allowing for more reliable trial coating using a coating liquid having the same components as the coating liquid supplied to the wells 9A of the first substrate in the main coating.

[0046] The XY coordinates of the applied first coating liquid are corrected (S3-2). FIG. 12 is a schematic diagram showing the XY coordinates of the trial-applied first coating liquid and the XY coordinates of its target position. As shown in FIG. 12, the target area to which the first coating liquid is to be trial-applied is indicated by R1. Assuming that the first coating liquid is circular, the position of the center of the circle is indicated by Ro1. The area to which the first coating liquid is actually trial-applied is indicated by R2. The position of the center of the actually applied first coating liquid is indicated by Ro2. Positional deviations (errors) occur between R1 and R2, and between Ro1 and Ro2. This positional deviation is corrected. At this time, the positional deviation may be corrected using the outlines of the outermost edges of R1 and R2 and the center positions Ro1 and Ro2.

[0047] 11 again, a first measurement step is performed in which the diameter of the trial-applied first coating liquid is measured (S3-3). Specifically, if the first coating liquid is circular in plan view from the Z direction, the diameter of the circle is determined. If the first coating liquid is non-circular in plan view from the Z direction, a circle having the same area as the trial-applied first coating liquid in plan view from the Z direction is assumed. The diameter of the assumed circle is considered to be the diameter of the trial-applied first coating liquid.

[0048] Next, a first determination step is performed to determine whether or not a change in the push-in amount is necessary (S3-4). Specifically, in the first determination step, it is determined whether or not a change in the first push-in amount used when applying the first application liquid in step (S3-1) is necessary. A first difference, which is the difference between the diameter of the first application liquid obtained in the first measurement step and a target diameter, which is a target value for that diameter, is calculated. Based on this first difference, it is determined whether or not a change in the first push-in amount is necessary.

[0049] 13 is a schematic diagram illustrating the relationship between an area where the first coating liquid should be trial-applied and an area where the first coating liquid is actually applied, for explaining the first determination step (S3-4). As shown in FIG. 13, the first coating liquid to be trial-applied is circular in plan view from the Z direction. The area (diameter) where the first coating liquid should be applied is indicated by R1. The diameter of R1 is the target value (target diameter) of that diameter. In contrast, the first coating liquid that is actually applied is indicated by R2. If the actual first coating liquid is non-circular, R2, which is assumed to be a circle with the same planar area, is illustrated.

[0050] The target diameter R1 can also be stored in a device provided in the coating apparatus 100. However, the target diameter R1 is basically a numerical value that is determined and stored by an operator.

[0051] In the case of A in Figure 13, the actual coated area R2 is smaller than the target diameter indicated by R1. In this case, the push-in amount in the next trial coating is increased compared to the immediately preceding trial coating. On the other hand, in the case of B in Figure 13, the actual coated area R2 is larger than the target diameter indicated by R1. In this case, the push-in amount in the next trial coating is decreased compared to the immediately preceding trial coating.

[0052] The target diameter is not a single diameter value but is expressed as a range of allowable diameter values. For example, the target diameter is set to a range of ±10% centered around 1000 μm. In other words, in this example, the target diameter is set to approximately 900 μm or more and 1100 μm or less.

[0053] The process up to this point (from step (S3-1) to step (S3-4)) is referred to as the first trial coating. The subsequent processes (step (S3-5) and thereafter, which will be described next) are referred to as the second trial coating.

[0054] Referring again to FIG. 11 , if R2 is within the range of the target diameter R1 in step (S3-4) and a second trial application is not required, the process proceeds from step (S3-4) in the direction indicated by the arrow "NO." In other words, the trial application process (S3) of the first coating liquid is terminated. However, if R2 is outside the range of the target diameter R1 in step (S3-4) as shown in FIG. 13 , it is determined in the first determination step that the first push-up amount needs to be changed and a second trial application is required. In this case, after the first trial application process, the process proceeds from step (S3-4) in the direction indicated by the arrow "YES." Then, as shown in FIG. 11 , the first coating liquid is again applied by a push-up amount different from the previous application (S3-5). At this time, a second trial application of the first coating liquid is performed on the application portion of the bottom surface 92 of one well 9A by a second push-up amount different from the first push-up amount, taking into account the first difference.

[0055] A second measurement step (S3-6) is performed to measure the diameter of the first coating liquid applied in the step (S3-5) of re-applying the first coating liquid. The method for determining this diameter is the same as in the first measurement step (S3-3). A determination step (S3-7) is performed to determine whether the diameter of the first coating liquid obtained in the second measurement step (S3-6) is within a target diameter range.

[0056] If the determination step (S3-7) determines that R2 (see FIG. 13) is within the range of the target diameter R1 and thus does not require a second trial application, the process proceeds from step (S3-7) in the direction indicated by the arrow "YES." In other words, the trial application step (S3) of the first application liquid is terminated. However, if the determination step (S3-7) determines that R2 (see FIG. 13) is outside the range of the target diameter R1, it is determined that the second push-in amount needs to be changed and a second trial application needs to be performed. In this case, the process proceeds from step (S3-7) in the direction indicated by the arrow "NO." Then, as shown in FIG. 11, the first application liquid is applied again using a push-in amount different from the previous amount (S3-5). The second trial application step, i.e., steps (S3-5) through (S3-7), is repeated until the diameter R2 (see FIG. 13) of the first application liquid obtained in the second measurement step (S3-6) falls within the range of the target diameter R1.

[0057] In the second trial application process described above, the coating may be applied to the same well 9A as in the first trial application process. In the second trial application process, the coating may be applied to a different well 9A than in the first trial application process. When the coating is applied to the same well 9A as in the first trial application process in the second trial application, the coating is applied to a location that does not overlap with a location coated in a previous process (such as the first trial application), and is applied to an empty space that has not yet been coated.

[0058] When the second trial coating step is repeated multiple times, the first coating liquid is re-applied with a second push-in amount that is different from the immediately preceding second push-in amount (S3-5). Thus, when the second trial coating step is performed multiple times, the second push-in amount in each iteration is different from one another. In other words, multiple different second push-in amounts are assumed. In each second trial coating, the difference between the diameter of the coating liquid obtained in the immediately preceding trial coating step and the target diameter (the first difference if the immediately preceding trial coating step was the first trial coating) is taken into consideration.

[0059] From the above, the second trial coating step can be said to be as follows, regardless of the number of times it has been performed: In the second trial coating step, in the case of the first substrate, the first coating liquid is again applied to the coating portion of one well 9A using a different push-in amount from the push-in amount used in the previous coating step, depending on the difference between the diameter at the time of the previous coating step (first trial coating or second trial coating) and the target diameter (S3-5).

[0060] When the trial application (S3) of the first coating liquid in FIG. 11 is performed on a second substrate different from the first substrate, the same process as the first trial application is performed on the coating surface (coating portion) of the second substrate. It may be determined that the first indentation amount needs to be changed during the process of performing the same process as the first trial application using the second substrate. In that case, the same process as the second trial application is further performed on the second substrate. In this case, the first trial application and the second trial application are not necessarily performed on the bottom surface (coating surface) of the well 9A. This is because the second substrate is not limited to a multi-well plate and may be a slide glass or the like. The same applies to the next step (S4). As described above, the application amount of the first coating liquid, etc., is adjusted.

[0061] 11, the process (S3) using the first substrate and the process (S3) using the second substrate are generally similar. Regardless of whether the process is for the first substrate or the second substrate, the process (S3) may be hereinafter referred to as (c).

[0062] Referring again to FIG. 6 , a trial supply of the second coating liquid is performed (S4). This is a step in which the second coating liquid is trial-applied (trially dropped) onto the substrate from the dropping mechanism 105 onto the application portion of at least one well 9A of the first substrate prior to the actual application for supplying the cell tissue to the first substrate. However, like the first coating liquid, the second coating liquid may also be applied from the needle application mechanism 104 of FIG. 1 . Because the second coating liquid may be supplied by either application or dropping, the second coating liquid may sometimes be referred to as "supplied" here. However, to clarify that it is on the same level as the first coating liquid, hereinafter, the bioink supplied after the first coating liquid will be referred to as the second "coating liquid," regardless of whether it is applied or dropped.

[0063] Step (S4) is performed on the same coating surface as the position where the first coating liquid was trial-coated in step (S3). When step (S4) is performed on the first substrate, the second coating liquid is supplied into the coating portion of the same well 9A as the well 9A where step (S3) was performed. When step (S4) is performed on the second substrate, the second coating liquid is supplied to the same position on the coating surface of the flat plate member as the position where the first coating liquid was trial-coated.

[0064] It is preferable that the second coating liquid is supplied directly above the first coating liquid applied in step (S3). That is, it is preferable that the second coating liquid is supplied so as to cover the first coating liquid. However, the second coating liquid may be supplied to a position other than the supply position of the first coating liquid. In this case, the second coating liquid is supplied so as not to cover the first coating liquid.

[0065] 14 is a flowchart of the trial supply of the second coating liquid (S4). As shown in FIG. 14, in the trial supply of the second coating liquid (S4), the second coating liquid is first supplied under first conditions (S4-1). The first conditions are the discharge position, discharge time, and discharge pressure of the second coating liquid when supplied to the coating surface. Here, the discharge position of the second coating liquid refers to the position in the vertical direction (Z direction) of the tip of the dripping mechanism 105 from which the second coating liquid is discharged.

[0066] The second coating liquid preferably contains a thickener and a solvent as its main components, and by doing so, the shape retention of the first coating liquid can be improved by covering the first coating liquid with a thickener having a high viscosity.

[0067] The XY coordinates of the supplied second coating liquid are corrected (S4-2), in the same manner as in the step (S3-2) for the first coating liquid shown in FIGS.

[0068] Referring again to FIG. 14 , next, a second difference between the output value under the first condition during trial supply of the second coating liquid and the target output, which is the target value for the first condition, is calculated (S4-3). Specifically, the actual discharge position, discharge time, and discharge pressure of the second coating liquid in the step (S4-1) of trial supplying the second coating liquid are obtained as the output value under the first condition. Typically, there is an error between these and the target values, which are the discharge position, discharge time, and discharge pressure values ​​input when performing step (S4-1). This error is calculated as the second difference. The discharge position is obtained, for example, by measuring the Z coordinate of the tip of the dripping mechanism 105. The output value for the discharge time and the output value for the discharge pressure are confirmed by investigating whether or not any of the second coating liquid has not been supplied within the required operating time. This results in the output value for the discharge time and the output value for the discharge pressure. However, as with the first coating liquid, the diameter of the supplied second coating liquid may also be examined as the target output (output value), which is the target value. The diameter of the second liquid can be measured by optimizing the lens magnification of the observation optical system. In other words, it is not necessary to check the output values ​​of the discharge time and the discharge pressure as the target output.

[0069] Based on the second difference thus obtained, it is determined whether or not the first condition, such as the discharge time, needs to be changed. This step is referred to as the second determination step (S4-4). Specifically, if the output value of the first condition is outside the target value range and smaller than the target value, the input value is increased so that the output value of the first condition becomes larger. Conversely, if the output value of the first condition is outside the target value range and larger than the target value, the input value is decreased so that the output value of the first condition becomes smaller.

[0070] The process up to this point (from step (S4-1) to step (S4-4)) is referred to as the first trial supply. The process thereafter (step (S4-5) and thereafter, which will be described next) is referred to as the second trial supply.

[0071] If the output value of the first condition in step (S4-4) is within the target value range and another trial supply is not required, the process proceeds from step (S4-4) in the direction indicated by the arrow "NO." In other words, the process (S4) of trial supplying the second coating liquid is terminated. However, if the output value of the first condition in step (S4-4) is outside the target value range, it is determined in the second judgment step that the first condition must be changed and another trial supply must be performed. In this case, the process proceeds from step (S4-4) in the direction indicated by the arrow "YES." Then, the second coating liquid is again supplied under conditions different from those immediately before (S4-5). At this time, a second trial supply of the second coating liquid is performed on the coating portion of one well 9A under second conditions, such as discharge conditions different from the first conditions, while taking into account the second difference. In the second trial supply, it is preferable that the second coating liquid be supplied so as to cover the first coating liquid.

[0072] In the step of supplying the second coating liquid again (S4-5), a step of checking the output value under the conditions when the second coating liquid was supplied (S4-6) is performed. It is then examined whether the output value under other conditions different from the first conditions in the step of supplying the second coating liquid again is within the range of the target output (S4-7).

[0073] If the output value of the condition (other condition different from the first condition) in the examination step (S4-7) is within the target value range and another trial supply is not required, the process proceeds from step (S4-7) in the direction indicated by the YES arrow. In other words, the trial supply step (S4) of the second coating liquid is terminated. However, if the output value of the condition in step (S4-7) is outside the target value range, it is determined that the second condition needs to be changed and another trial supply is required. In this case, the process proceeds from step (S3-7) in the direction indicated by the NO arrow. Then, as shown in FIG. 11 , the second coating liquid is again supplied (S4-5) under conditions different from those immediately before. The second trial supply step, i.e., steps (S4-5) through (S4-7), is repeated until the output value of the other condition (such as the ejection position of the second coating liquid) obtained in the examination step (S4-7) is within the target output range.

[0074] When the second trial supply step is repeated multiple times, the first application liquid is re-applied under second conditions that are different from the immediately preceding second conditions (S4-5). Thus, when the second trial supply step is performed multiple times, the second conditions are different for each step. In other words, multiple different amounts are assumed as the second conditions. In each second trial supply, the difference between the output value of the condition in the immediately preceding trial supply step and the target value of that condition (the second difference if the immediately preceding trial supply step was the first trial supply) is taken into consideration.

[0075] From the above, the second trial supply step can be described as follows, regardless of the number of times it has been performed. In the second trial supply step, in the case of the first substrate, the second coating liquid is again supplied to the coating portion of one well 9A under conditions different from those used during the previous supply, depending on the difference between the conditions (output value) under the previous supply (first trial supply or second trial supply) and the target output, which is the target value for those conditions (S4-5). The target output here may be the discharge position, discharge time, and discharge pressure of the second coating liquid, or it may also be the diameter of the second coating liquid.

[0076] When the trial supply (S4) of the second coating liquid in FIG. 14 is performed on a second substrate different from the first substrate, the same process as the first trial supply is performed on the coating surface (coating section) of the second substrate. It may be determined that the first conditions need to be changed during the process of performing the same process as the first trial supply using the second substrate. In that case, the same process as the second trial supply is further performed on the second substrate. In this case, the first trial coating and the second trial coating are not necessarily performed on the bottom surface (coating surface) of the well 9A. This is because the second substrate is not limited to a multi-well plate and may be a glass slide or the like. In this manner, the amount of the second coating liquid dispensed is adjusted.

[0077] 14, the process (S4) using the first substrate and the process (S4) using the second substrate are generally similar. Regardless of whether the process is for the first substrate or the second substrate, the process (S4) may be hereinafter referred to as (d).

[0078] The above description is based on the assumption that the steps prior to the main coating (S5) are performed in the order of (a), (b), (c), and (d). While this order is acceptable, the order of the steps may be changed. When performing trial coating and trial supply on a first substrate, the processes may be performed in the following order. For example, the order may be (b), (a), (c), and (d). Alternatively, the order may be (a), (b), (d), and (c), or (b), (a), (d), and (c). When performing trial coating and trial supply on a second substrate, the order may be, for example, (b), (a)', (c), (d), and (a), or (b), (a)', (d), (c), and (a). Alternatively, the order may be (a), (b), (a)', (c), and (d), or (a), (b), (a)', (d), (c), and (a). Alternatively, the order may be (a), (b), (a)', (c), and (d), or (a), (b), (a)', (d), and (c).

[0079] Referring again to FIG. 6, after the trial application and trial supply performed prior to the actual application are completed, the actual application is performed (S5). That is, after the diameter of the first application liquid is adjusted to within the target diameter range, the application liquid for producing cellular tissue is supplied to the application portions of the well 9A of the first substrate other than the application portions subjected to the trial application (S3) and trial supply (S4). The application liquid here refers to both the first application liquid and the second application liquid. This allows the desired cellular tissue to be produced.

[0080] (Embodiment 2) In this embodiment, the process is basically the same as in Embodiment 1. Therefore, the description of the portions of this embodiment that overlap with Embodiment 1 will not be repeated. In Embodiment 1, when performing trial coating or the like using a multiwell plate as the first substrate, for example, no consideration is given to which of the multiple wells 9A (see FIG. 3) included in plate 8 the trial coating is to be performed on. However, typically, trial coating or the like is performed on some of the multiple wells 9A in plate 8, and actual coating is performed on the other wells 9A. In this embodiment, a method will be described in which the depth of only some of the multiple wells 9A is measured and the depth of the other wells 9A is calculated from the measured depth.

[0081] Figure 15 is a schematic diagram showing an example of well selection for measuring bottom depths among wells contained in a multiwell plate serving as a first substrate. As shown in Figure 15, plate 8 is a multiwell plate with 8 vertical rows and 12 horizontal columns. The vertical rows of wells are designated A to H. The horizontal columns of wells are designated 1 to 12. Figure 15 shows a total of 12 types of well selection patterns (1) to (12) for measuring bottom depths (positions in the Z direction). Wells selected for measuring bottom depths are hatched. Hereinafter, for example, a well at the intersection of row A and column 1 may be designated as "A1."

[0082] The wells of the multiwell plate selected for measuring the Z-direction position of the bottom surface are not particularly limited. In FIG. 15 , for example, a single arbitrarily selected well, such as A1 in (1), may be selected. Alternatively, the wells may be the two diagonally opposite outermost corner wells, such as A1 and H12 in (2). Alternatively, the wells may be the four outermost corner wells, such as A1, H1, A12, and H12 in (3). Alternatively, a total of nine wells may be selected, including the four outermost corner wells, the center (D6), and the centers of the four outermost sides, as in (4). These nine wells are arranged at approximately equal intervals. Alternatively, all wells in column 1 may be selected, as in (5). Alternatively, all wells in row A may be selected, as in (6). Alternatively, all wells in both column 1 and row A may be selected, as in (7). Alternatively, all wells in columns 1, 12, row A, and row H around the entire circumference may be selected, as in (8). As in (9), all wells in rows A, C, E, and G may be selected. As in (10), all wells in columns 1, 3, 5, 7, 9, and 11 may be selected. As in (11), wells may be selected in a checkerboard pattern. As in (12), all wells may be selected. Alternatively, the above (1) to (11) may be combined as appropriate. For example, as a modification of (2), wells may be selected in a diagonal direction, such as A1, B2, C3, and D4. As a modification of (10), all wells in columns 1, 3, 5, 7, 8, 10, and 12 may be selected. The measured well depth values ​​are stored.

[0083] Except for the case of (12) in Figure 15, the depths of some wells are not measured. Except for the case of (1) in Figure 15, the depths of multiple wells are measured. When the depths of at least two wells are measured, as in (2) to (11) in Figure 15, the depth of the coating surface of the unmeasured wells is estimated by the following calculation of the first method.

[0084] Fig. 16 is a schematic diagram illustrating a method for estimating the bottom depth of wells whose depths have not been measured. In Fig. 16, five wells numbered 1 to 5 are assumed to be aligned, for example, in the Y direction (the horizontal direction in Fig. 15). In Fig. 16, it is assumed that the bottom depths of wells 1, 3, and 5 have been measured (scanned). In Fig. 16, the bottom depths of wells 2 and 4 have not been measured.

[0085] As shown in FIG. 16 , well 2, whose depth has not been measured, is located between the positions of wells 1 and 3, whose depths have been measured, in the Y direction. In this case, in the process of estimating the bottom depth of well 2, the ratio of the distances in the Y direction between each of the two wells, well 1 and well 3, whose depths have been measured, and well 2, whose depth is to be estimated, is calculated. The ratio of the distances in the Y direction is determined by considering the distance between the centers of the wells when viewed in a plane from the Z direction. This is particularly preferable when the wells have a circular shape when viewed in a plane from the Z direction. In this case, the ratio of the distances is calculated as the division ratio. The division ratio means an internal division ratio or an external division ratio. In the example of FIG. 16 , well 2 is located between well 1 and well 3 in the Y direction. Therefore, well 2 internally divides the line connecting well 1 and well 3. Based on this division ratio (internal division ratio), the depth of well 2 is calculated from the measured depth values ​​of wells 1 and 3.

[0086] In calculating the depth of well 2, an estimated value of the depth of well 2 is obtained under the assumption that the depth changes at the same rate between well 1 and well 3. Specifically, the depth values ​​of wells 1 to 3 are indicated on the vertical axis (well bottom height) in Figure 16. Well 2 is located midway between wells 1 and 3 in the Y direction. In other words, well 2 divides wells 1 and 3 internally at a 1:1 ratio. The bottom height of any well between well 1 and well 3 can be determined from the height direction position of a line connecting well 1 and well 3 (at their centers). This is because the bottom height (depth) changes at the same rate between well 1 and well 3, and therefore the slope of the line connecting well 1 and well 3 is constant. In this example, if the bottom height of well 1 is 100 μm and the bottom height of well 3 is 0 μm, the bottom height of well 2 is 50 μm. Therefore, the bottom height of well 2 can be predicted from the slope of the line connecting well 1 and well 3 in Figure 16. Similarly, the bottom height of well 4 can be predicted from the slope of the line connecting well 3 and well 5. For example, if well 4 is located at a position that divides well 3 and well 5 internally at a ratio of 1:2, and the bottom height of well 5 is 60 μm, then the bottom height of well 4 is 20 μm.

[0087] The above can be expressed as a formula, as in the following equation (1). Let h(r) be the bottom height of well r in Figure 16, whose bottom height is to be estimated. Let h(m) be the measured bottom height of well m, and h(n) be the measured bottom height of well n that is different from well n. Wells m, r, and n are arranged so that they are on a straight line in rows A to H or columns 1 to 12 in Figure 15. The magnitude relationship is m<r<n. For example, in Figure 16, wells m, r, and n are wells 1, 2, and 3, or wells 3, 4, and 5. The value h(r) to be found in this case is

[0088]

[0089] The second term, h(n), in the above equation (1) may be h(m). Alternatively, the depth of the coating surface of unmeasured wells may be estimated by the calculation of the following second method. FIG. 17 is a schematic diagram showing the arrangement of 96 wells contained in a plate. FIG. 17 schematically shows the plate of FIG. 3, which has basically the same configuration as FIG. 15. Therefore, a detailed description of FIG. 17 will be omitted. FIG. 18 is a graph showing the measured bottom height of each well in the plate of FIG. 17. In both (A) and (B) of FIG. 18, the horizontal axis represents columns 1 to 12 of the plate shown in FIG. 17, and the vertical axis represents the measured bottom height of the well. Each broken line is shown for each row of the plate of FIG. 17. (A) of FIG. 18 shows the trend in the bottom depth of wells contained in plates manufactured by a certain manufacturer. (B) of FIG. 18 shows the trend in the bottom depth of wells contained in plates manufactured by a different manufacturer than (A).

[0090] The second method is applicable when the bottom height of only one well A1 is measured, as shown in FIG. 15 (1). As a specific example, consider the use of a plate manufactured by a manufacturer that displays the data shown in FIG. 18 (A). First, data indicating the depths of the bottoms of 96 wells, as shown in FIG. 18 (A), is stored in the storage medium of the coating device 100. In the data shown in FIG. 18 (A), for example, the bottoms of the wells in columns 1 and 12 tend to be high, while the bottoms of the wells in column 6 tend to be low. Furthermore, the bottoms of the wells in rows A, B, and C tend to be high, particularly in columns 6 to 12.

[0091] Next, as shown in Figure 15 (1), the bottom height of only well A1 of the plate is actually measured. The bottom height of each well of the plate other than well A1 is calculated as follows: The difference between the actual measurement value of well A1 and the value of well A1 in the stored data is calculated. Taking this difference into account, and assuming that the heights of each well other than well A1 will increase or decrease in the same manner as the stored data, the heights of each well other than well A1 are estimated.

[0092] For example, consider a case where the measured bottom height of well A1 of the plate to be measured is 10 μm. In FIG. 18A, the bottom height of well A1 is 0 μm. Therefore, the bottom position of the well of the plate to be measured is considered to be 10 μm higher than the data in FIG. 18A. Therefore, for example, the bottom height of well A7 of the plate is estimated to be -90 μm, 10 μm higher than the -100 μm shown in FIG. 18A.

[0093] Furthermore, the depth of the coating surface of unmeasured wells may be estimated by calculation according to the following third method: In the third method, the depth of the bottom surface may be estimated by calculation using artificial intelligence.

[0094] By using any of the above-described first to third methods, the bottom height (depth) of the wells 2 and 4 is determined (estimated). Based on the estimated value, the pushing amount of the coating needle 24 into the wells 2 and 4 is determined. The pushing amount here is the pushing amount of the coating needle 24 in the trial coating of the first coating liquid (S3) in the first embodiment, etc.

[0095] (Operations and Effects) The operations and effects of the first and second embodiments will be described below, while touching upon the background art and problems that led to the present embodiment as appropriate.

[0096] In the method for producing cellular tissue according to the present disclosure, a first substrate (plate 8) for forming cellular tissue, having a plurality of wells 9A, is placed in equipment. The depth of the application surface of the first substrate (bottom surface 92 of wells 9A) relative to the surface 8B of the first substrate is measured. Prior to supplying cellular tissue to the first substrate, a first trial application step is performed in which a first application liquid is applied to at least one application portion that is a portion of the application surface (bottom surface 92) of the first substrate. In the first trial application step, the first application liquid is applied to one application portion (part of the bottom surface 92) with a first depression amount of the application needle 24 corresponding to the depth. A first measurement step is performed in which the diameter of the first application liquid is measured.

[0097] As shown in Figure 18, there is variation in the bottom height of multiple wells 9A formed in one plate 8. The upper and lower graphs in Figure 18 show different trends. In other words, the trend in the variation in bottom height of wells 9A is not uniquely determined and may vary from manufacturer to manufacturer. Therefore, the depth of the coating surface of each (at least one) well 9A of the plate 8 to be used is measured. This allows the variation in the bottom height of the wells of the plate 8 to be determined in advance. This allows the insertion amount of the coating needle to be adjusted while taking into account the bottom height of each well 9A. This reduces variation in the coating amount (diameter) of the first coating liquid among multiple wells 9A. For example, it is possible to prevent the first coating liquid in states A and B in Figure 13 from being mixed within a single plate 8.

[0098] However, simply grasping the bottom height of the multiple wells 9A formed in the plate 8 is insufficient. Figure 19 is a schematic diagram showing variations in the amount of first coating liquid adhered to the coating needles. As shown in Figure 19, even if the state of the first coating liquid is the same and the heightwise position of the plate 8 is the same, the amount of first coating liquid adhered to the coating needles 24 may vary. Specifically, environmental conditions such as temperature and humidity vary, particularly between different days of work. Therefore, even if the first coating liquid is in the same state, the adhesion state of the first coating liquid to the coating needles 24 varies. The coating needle 24A on the left side of Figure 19 has a larger amount of first coating liquid A adhered to it than the coating needle 24B on the right side. The distance between the first coating liquid A on the coating needle 24A side and the surface 8B (coating surface) of the plate 8 is defined as h1. The distance between the first coating liquid A on the coating needle 24B side and the surface 8B (coating surface) of the plate 8 is defined as h2. h1 is shorter than h2 by h. Therefore, if coating needle 24A and coating needle 24B are pressed in by the same amount, coating needle 24A will apply a larger amount of first coating liquid A. In other words, even if the bottom height of the well is measured and the coating needle is pressed in based on that result, a difference in the amount applied will occur, as shown in A and B in Figure 13.

[0099] Therefore, in this embodiment, a first trial application of the first coating liquid is performed in advance. The first application is performed with a first push-in amount corresponding to the measured depth of the coating surface of the well 9A or the like, and the diameter of the applied first coating liquid is measured. This reduces the environmental variation that inevitably occurs between work days, and allows the amount of push-in of the coating needle to be determined in advance with high precision for each well bottom height.

[0100] The first coating liquid A is deposited on the coating needle 24 each time the coating needle 24 is pressed into the substrate to apply the coating liquid. In other words, the number of times the first coating liquid A is deposited on the coating needle 24 is equal to the number of times the coating needle 24 is pressed into the substrate to apply the coating liquid. Variation in the amount of the first coating liquid A deposited on the coating needle 24 is negligibly small within the same day. Therefore, it is preferable that the trial coating process be performed immediately before the actual coating operation on that day. If the relationship between the well height and the coating amount during the operation can be understood immediately before the actual coating operation, variation in the coating amount between coating portions, such as the multiple wells 9A formed on the plate 8, during the actual coating operation can be reduced. As a result, the coating liquid can be supplied stably during the actual coating operation on that day.

[0101] In the method for producing cell tissue, the first trial application step further includes a first determination step. In the first determination step, a first difference between the diameter of the first application liquid obtained in the first measurement step and a target diameter, which is a target value for the diameter, is calculated, and whether or not the first depression amount needs to be changed is determined based on the first difference. If it is determined in the first determination step that the first depression amount needs to be changed, a second trial application step of applying the first application liquid to one application site at a second depression amount different from the first depression amount is further performed after the first trial application step.

[0102] The penetration amount is adjusted as needed by a second trial application based on the diameter of the first application liquid obtained in the first measurement step, thereby reducing environmental variations between different work days and further enhancing the effectiveness of predetermining the penetration amount of the application needle for each well bottom height.

[0103] In the above-described method for producing cell tissue, the second trial application step involves reapplication of the first coating liquid to one application area using a different pressing amount than the pressing amount used in the previous application, depending on the difference between the diameter at the previous application and the target diameter. A second measurement step is performed to measure the diameter of the first coating liquid applied in the reapplication step of the first coating liquid. A determination step is performed to determine whether the diameter of the first coating liquid obtained in the second measurement step is within the target diameter range. The second trial application step is repeated until the diameter of the first coating liquid obtained in the second measurement step falls within the target diameter range. In this way, the optimal pressing amount can be determined based on the bottom height of the well for that work day. This reduces variation in the amount of coating between application areas, such as multiple wells 9A formed on the plate 8.

[0104] In the above-described method for producing cell tissue, the first substrate is preferably a multi-well plate (plate 8) having a plurality of wells 9A, and the application surface is preferably the bottom surface 92 of each of the plurality of wells 9A. In this way, trial application can be performed on a plurality of wells 9A with different bottom heights. This allows for more accurate control of the amount of pressure to be applied to each individual well 9A.

[0105] In the method for producing cell tissue described above, in the depth measuring step, the depth of at least one well 9A among the multiple wells is measured. The depths of the coating surfaces (bottom surfaces 92) of other wells 9A included in the first substrate, other than the at least one well 9A measured in the depth measuring step, relative to the surface 8B of the first substrate are estimated by calculation. The amount of depression for the other wells is determined based on the depths of the other wells 9A estimated in the depth estimating step.

[0106] Reducing the number of wells 9A whose depths are measured has the advantage of shortening the work time. On the other hand, if the depth of an unmeasured well 9A is randomly assumed to be the same as that of other adjacent wells 9A without relying on calculation, the error from the actual depth will increase. In other words, the accuracy of the assumed bottom height of the well 9A will decrease. Conversely, measuring the depth of, for example, all wells 9A has the advantage of being able to grasp the bottom height of the well 9A with high accuracy. On the other hand, measuring all wells 9A has the disadvantage of lengthening the work time. Therefore, from the perspective of compensating for both disadvantages, the depth of only some wells 9A is actually measured, and the depth of the other wells 9A is estimated by calculation. This shortens the work time while accurately grasping the depth of more (all) wells 9A.

[0107] In the method for producing the cell tissue, at least two wells 9A are measured for depth among the plurality of wells. The other wells 9A are positioned between the positions of at least two of the plurality of wells 9A for which depths are measured. In the depth estimation step, the depth of the other wells 9A is calculated from the depths of the two wells 9A measured in the depth measurement step based on a division ratio, which is the ratio of the distance between each of the two wells 9A for which depths are measured and the other wells 9A for which depths are to be estimated. In the calculation, depth estimates are obtained under the assumption that the depths change at the same rate between the two wells 9A. This reduces the work time and allows the depths of more (all) wells 9A to be determined with high accuracy.

[0108] The above-described method for producing cell tissue preferably further includes a step of storing the measured depths in the depth measurement step. This allows the depths of unmeasured wells 9A to be estimated by calculation from the stored measured depth values ​​of the wells 9A. Furthermore, when performing a subsequent coating step on wells 9A whose depths have been measured and stored, the highly accurate measured and stored values ​​can be used to control the amount of indentation with high precision. This effectively reduces variation in the amount of coating.

[0109] In the method for producing cellular tissue, prior to supplying cellular tissue to a first substrate, a first trial supply of the second coating liquid is performed to the coating portion of at least one well 9A of the first substrate under first conditions, which include a discharge position, a discharge time, and a discharge pressure of the second coating liquid. In the first trial supplying step, the second coating liquid is supplied to the coating portion of one well 9A. A second determination step is performed in which a second difference between the output value under the first condition in the second coating liquid supplying step and a target output, which is a target value for the first condition, is calculated, and whether or not the first condition needs to be changed is determined based on the second difference. If it is determined in the second determination step that the first condition needs to be changed, a second trial supply of the second coating liquid to the coating portion of one well 9A under second conditions different from the first conditions is further performed.

[0110] In the trial supply of the second coating liquid, the process of controlling the output value is performed in a manner basically similar to that of the trial supply of the first coating liquid. This achieves the same effect as the trial supply of the first coating liquid. In other words, it is possible to reduce the variation in the supply amount of the second coating liquid.

[0111] In addition, with regard to the dropping of the second coating liquid, the height position of the coating surface, such as the bottom of the well, has less effect on the dropping state compared to the example in which the coating needle is pressed to apply the first coating liquid. However, adjusting the Z-direction ejection position of the second coating liquid is important for controlling the dropping state of the second coating liquid. For this reason, the ejection position is adjusted. In addition, the ejection time and ejection pressure are also important for controlling the dropping state of the second coating liquid, and are therefore adjusted.

[0112] In the above-described method for producing cell tissue, the second trial supply step involves resupplying the second application liquid to the application portion of one well 9A under different conditions for the discharge position, discharge time, and discharge pressure used in the previous supply, depending on the difference between the target output and the target output for the conditions of the discharge position, discharge time, and discharge pressure of the second application liquid in the previous supply. It is then examined whether the output value for the other conditions in the resupply step of the second application liquid is within the target value range. The second trial supply step is repeated until the output value for the other conditions in the examination step falls within the target value range. This reduces variation in the amount of second application liquid supplied.

[0113] In the method for producing cell tissue, a second substrate different from the first substrate is placed in the equipment. The height position of the coating surface of the second substrate to which the first coating liquid is applied is measured. A process similar to the first trial coating is performed on the coating surface of the second substrate. If it is determined that the first depression amount needs to be changed in the process of performing a process similar to the first trial coating using the second substrate, a process similar to the second trial coating is further performed on the second substrate.

[0114] The trial application (S3) of the first application liquid is not limited to the first substrate for cell tissue formation, and may be performed on a second substrate unrelated to the first substrate. As described above, the amount of the first application liquid attached to the application needle 24 is constant within the same day as the day on which the actual application is performed. Therefore, within the same day, the amount of application by pressing is a function only of the Z-direction position (height) of the application surface. Therefore, as long as the Z-direction height of the application surface is known by measurement, any substrate unrelated to the actual application can be used for the trial application. This increases the options for substrates to be used for trial application (trial application).

[0115] If the amount of coating after pressing were determined solely by the height of the well bottom (if the amount of adhesion to the coating needle was unrelated to the amount of coating), trial coating using a second substrate would be meaningless. However, as described above, in reality, the amount of coating after pressing is determined by the height of the well bottom (coating surface) and the amount of adhesion to the coating needle 24, which varies from work day to work day. Therefore, even when using a second substrate that is unrelated to the actual coating and for which the height position of the coating surface has been measured, the relationship between the height of the coating surface and the amount of coating can be derived with high precision, taking into account the daily variation in the amount of adhesion to the coating needle 24 on a given day.

[0116] Therefore, when the actual coating is performed using the first substrate, coating with reduced variation can be achieved simply by checking the height of the well bottom (coating surface) where the actual coating is performed. If the relationship between the height of the coating surface of the second substrate and the coating amount (diameter) after pressing can be derived from the trial coating using the second substrate, this relationship can also be used during the actual coating using the first substrate.

[0117] The method for producing cell tissue further includes a step of performing a process similar to the first trial supply on the coating surface of the second substrate. If it is determined that the first condition needs to be changed in the step of performing a process similar to the first trial supply using the second substrate, a process similar to the second trial supply is further performed on the second substrate. As with the trial application of the first coating liquid, the trial supply of the second coating liquid is not limited to the first substrate, and a second substrate may also be used. As long as the height of the coating surface of the second substrate has been investigated, the same effect as when the first substrate is used can be obtained.

[0118] (Explanation of a Specific Manufacturing Method Using Diagrams) Here, an example using a multiwell plate is illustrated. However, similar procedures can be used when using equipment other than a multiwell plate, such as a dish or a glass slide. Figure 20 is a schematic diagram showing the state before the first coating liquid is applied in an embodiment. As shown in Figure 20, the tip of the coating needle 24 constituting the bioprinter is first immersed in the first coating liquid A (first bioink), and the first coating liquid A is attached to the tip of the coating needle 24. Figure 21 is a schematic diagram showing the composition of the first coating liquid in an embodiment. As shown in Figure 21, 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 Figure 21 include C1 and C2, which will be described later. Figure 22 is a schematic diagram showing the process of applying the first coating liquid in an embodiment. As shown in FIG. 22 , the tip of the coating needle 24 to which the first coating liquid A has been 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 coating needle 24 to which the first coating liquid A has been applied downward as indicated by arrow M1 in FIG. 20 . This causes the first coating liquid A to be applied to the bottom surface 92 of the well 9A. FIG. 23 is a schematic diagram showing the state after the first coating liquid has been applied in this embodiment. As shown in FIG. 23 , the coating needle 24 then moves upward as indicated by arrow M2. In this manner, the first coating liquid A is applied to a container such as the well 9A using a bioprinter of the so-called pin type (a coating needle type such as the needle coating mechanism 104 in FIG. 1 ).

[0119] Fig. 24 is a schematic diagram showing a step of supplying a second coating liquid in an embodiment. Fig. 25 is a schematic diagram showing the state inside a well after the step of Fig. 24 has been performed. As shown in Figs. 24 and 25, a second coating liquid B (second bioink) is supplied into well 9A so as to cover first coating liquid A applied to the inside of well 9A. The second coating liquid B may be dropped by a dispenser, for example, but the method of supplying 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 the dropping mechanism 105 of Fig. 1), and manual supply using a pipette.

[0120] 26 is a schematic diagram showing a step of supplying a culture medium in an embodiment. As shown in FIG. 26, 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.

[0121] FIG. 27 is a schematic diagram showing the state of cells in the first coating liquid before culture. FIG. 28 is a schematic diagram showing the state of cells in the first coating liquid after culture. As shown in FIGS. 27 and 28 (and FIG. 21), 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. 27 to the state shown in FIG. 28, and myocardial tissue is formed.

[0122] (Materials) The first coating liquid A is composed of cells to be cultured, a gel (a gel raw material if liquid) for preparing (producing) the cell tissue, and a first solvent. In addition to the above, various additives may be added to the first coating liquid A.

[0123] 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, gene modification, or gene recombination may be used. The cells may be derived from any animal (animal origin) including humans, mice, rats, and monkeys. The cells may be differentiated cells derived from stem cells. In other words, the cells may be differentiated cells derived from iPS cells or ES cells. The cells may be mesenchymal stem cells. The cells may be primary cells or established cell lines.

[0124] The type of cells 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. The first coating liquid A contains one or more of the above types of cells. In other words, the first coating liquid A may contain multiple types of cells.

[0125] 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 100 cells / mL.

[0126] The first coating liquid A does not necessarily contain a gel (a gel raw material before solidification: hereinafter referred to as a gel). For example, the first coating liquid A may contain either a natural polymer or a synthetic polymer. When a gel is used in the first coating liquid A, the material of the gel is not particularly limited. The gel in the 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.

[0127] 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 dedicated to various cells, PBS solution (+ or -), Tris buffer, and Tyrode's buffer.

[0128] 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, and ROCK. inhibitor, 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.

[0129] 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.

[0130] 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.

[0131] The medium for culturing cells, i.e., medium M (see FIG. 24 ) that is applied after second coating liquid B in the manufacturing 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 dedicated to various cells. These various types of medium M may be mixed in any ratio. Furthermore, various additives may 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.

[0132] The first substrate for culturing cells is, for example, a plate 8 having wells 9A. The plate 8 is not limited to the one having 96 wells 9A described above. The number of wells 9A in the plate 8 may be 6, 12, 24, 48, 96, 384, or 1536. The first substrate is not limited to a multi-well plate. The first substrate may be a circular dish of any diameter in plan view. Alternatively, the first substrate may be a microchannel, a slide glass, a cell disk LF, or a PDMS substrate. The bottom surface 92 of the wells 9A in the first substrate may be flat as shown in FIG. 4 or U-shaped as shown in FIG. 5. Although not shown, the bottom surface 92 may also be V-shaped.

[0133] The surface of the first substrate may be cell-adhesive, and the cell-adhesive first substrate may be formed from a cell-adhesive polymer material. Specifically, the cell-adhesive polymer material may be any one selected from the group consisting of collagen, gelatin, fibronectin, and Matrigel. Regardless of the type of the non-cell-adhesive first substrate, the contact angle of water (pure water) on the surface may be controlled by radiation. The low cell-adhesive first substrate is preferably formed using a low cell-adhesive polymer. Here, the low cell-adhesive polymer may be, for example, either an MPC polymer or P-HEMA. For example, a plate 8 may be formed in which the surface of the substrate is coated with either an MPC polymer or P-HEMA.

[0134] An experiment was conducted to actually form cell tissue based on the method for producing cell tissue of this embodiment. In this experiment, coating liquids were prepared as follows using an apparatus capable of supplying multiple coating liquids, such as coating apparatus 100 shown in Figure 1.

[0135] The plate 8 used was a multi-well plate having 96 wells 9A as shown in Figure 3. Droplets as described below were supplied into the wells 9A.

[0136] 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 thickener. The medium M was DMEM supplemented with 10% by mass of FBS.

[0137] 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 A was applied to one well 9A of plate 8. Next, a second coating liquid B was dripped onto the first coating liquid A to cover it. The second coating liquid B was dripped using a dispenser (see drip mechanism 105 in FIG. 1). Then, medium M was dripped onto the first coating liquid A to cover it. Medium M was dripped onto the first coating liquid A using a manual pipette. Cells were cultured in medium M to form a cell tissue. This process is as described in the section (Explanation of specific manufacturing method using diagrams).

[0138] Figure 29 is a phase-contrast microscope image taken immediately after application of the coating liquid in Example 1. During the production of the cell tissue shown in Figure 29, the depression amount of the coating needle 24 used to apply the first coating liquid was corrected as follows. The difference between the first diameter measurement (see S3-3 in Figure 11) performed using the first depression amount in the coating process of the first coating liquid (see S3-1 in Figure 11) and the tissue diameter was approximately 800 µm. Therefore, the depression amount was increased by 30 µm downward in the Z direction and re-coating was performed (see S3-5 in Figure 11). A second measurement of this diameter (see S3-6) revealed that the tissue diameter was approximately 1000 µm.

[0139] In this example, after the trial application, the actual application was performed on a total of 60 wells, from B2 to G11, of a 96-well plate, excluding one perimeter well. The diameter of the tissue applied to each well was 1062 ± 82 μm. The median diameter was within the range of the target diameter of 1000 μm ± 10%, and the half-width of the variation was less than 10% of the target diameter. Therefore, cell tissue of an appropriate diameter was stably formed.

[0140] (Supplementary Note) Various aspects of the present disclosure will be summarized below as supplementary notes.

[0141] (Supplementary Note 1) A method for producing cell tissue, comprising: a step of placing a first substrate for forming cell tissue within equipment; a step of measuring the depth of a coating surface of the first substrate relative to the surface of the first substrate; and a step of first trial applying a first coating liquid to at least one coating portion that is a part of the coating surface of the first substrate prior to supplying cell tissue to the first substrate, wherein the first trial application step includes a step of applying the first coating liquid to one coating portion with a first depression amount of an application needle corresponding to the depth; and a first measurement step of measuring the diameter of the first coating liquid.

[0142] (Appendix 2) The method for producing cell tissue described in Appendix 1, wherein the first trial application step further includes a first determination step of calculating a first difference between the diameter obtained in the first measurement step and a target diameter that is a target value of the diameter, and determining whether or not it is necessary to change the first pushing amount based on the first difference, and if it is determined in the first determination step that it is necessary to change the first pushing amount, a second trial application step of applying the first application liquid to one application portion with a second pushing amount that is different from the first pushing amount is further performed after the first trial application step.

[0143] (Appendix 3) The method for producing cell tissue described in Appendix 2, wherein the second trial application step includes: a step of re-applying the first application liquid to one application portion using a different pushing amount than the pushing amount used in the previous application, depending on the difference between the diameter in the previous application and the target diameter; a second measurement step of measuring the diameter of the first application liquid applied in the step of re-applying the first application liquid; and a determination step of determining whether the diameter of the first application liquid obtained in the second measurement step is within the range of the target diameter, and the second trial application step is repeated until the diameter of the first application liquid in the second measurement step falls within the range of the target diameter.

[0144] (Appendix 4) The method for producing cell tissue according to any one of Appendices 1 to 3, wherein the first application liquid contains cells, a gel raw material, and a solvent as main components.

[0145] (Supplementary Note 5) The method for producing cell tissue according to any one of Supplementary Notes 1 to 4, wherein the step of measuring the depth is performed by measurement using a laser sensor or measurement using autofocus of a camera.

[0146] (Appendix 6) The method for producing cell tissue according to any one of Appendices 1 to 5, wherein the first substrate is a multi-well plate having a plurality of wells, and the application surface is the bottom surface of each of the plurality of wells.

[0147] (Appendix 7) The method for producing cell tissue described in Appendix 6 further comprises the steps of: measuring the depth of at least one of the plurality of wells in the depth measuring step; estimating by calculation the depth of the coating surface of other wells included in the first substrate other than the at least one well measured in the depth measuring step relative to the surface of the first substrate; and determining the amount of depression for the other wells based on the depth of the other wells estimated in the estimation step.

[0148] (Appendix 8) A method for producing cell tissue as described in Appendix 7, wherein the method has at least two wells among the plurality of wells whose depths are measured, the other wells are positioned between positions where at least two of the plurality of wells whose depths are measured are positioned, and in the estimating step, the depth of the other well is calculated from the depths of the two wells measured in the depth measuring step based on a division ratio which is the ratio of the distance between each of the two wells whose depths are measured and the other well whose depth is to be estimated, and in the calculation, an estimated value of the depth is obtained under the assumption that the depth changes at the same rate between the two wells.

[0149] (Supplementary Note 9) The method for producing cell tissue according to any one of Supplementary Notes 1 to 8, further comprising the step of storing the depth measured in the step of measuring the depth.

[0150] (Appendix 10) A method for producing cell tissue according to any one of Appendices 1 to 9, comprising, prior to supplying cellular tissue to the first substrate, a step of performing a first trial supply of the second application liquid to at least one application portion of the first substrate under first conditions of a discharge position, a discharge time, and a discharge pressure of the second application liquid, wherein the first trial supply step includes: a step of supplying the second application liquid to one application portion; and a second determination step of calculating a second difference between an output value under the first condition in the step of supplying the second application liquid and a target output which is a target value for the first condition, and determining whether or not the first condition needs to be changed based on the second difference, wherein if it is determined in the second determination step that the first condition needs to be changed, a step of second trial supplying the second application liquid to one application portion under second conditions different from the first condition is further performed.

[0151] (Appendix 11) The method for producing cell tissue described in Appendix 10, wherein the second trial supplying step includes: a step of re-supplying the second application liquid to one of the application units under conditions different from the discharge position, discharge time and discharge pressure conditions used during the previous supply, depending on a difference between the target output and the conditions of the discharge position, discharge time and discharge pressure of the second application liquid during the previous supply; and a step of examining whether the output value under the other conditions in the re-supplying of the second application liquid is within the range of the target output, wherein the second trial supplying step is repeated until the output value under the other conditions in the examining step is within the range of the target output.

[0152] (Appendix 12) The method for producing cell tissue described in Appendix 10 or 11, wherein the output value of the discharge time and the output value of the discharge pressure are confirmed by checking whether or not the second application liquid has not been supplied within the required operating time.

[0153] (Appendix 13) The method for producing cell tissue according to any one of Appendices 10 to 12, wherein in the first trial supplying step and the second trial supplying step of the second coating liquid, the second coating liquid is supplied so as to cover the first coating liquid, and the second coating liquid is mainly composed of a thickener and a solvent.

[0154] (Appendix 14) A method for producing cell tissue according to any one of Appendices 10 to 13, further comprising the steps of: placing a second substrate different from the first substrate in equipment; measuring the height position of the coating surface of the second substrate onto which the first coating liquid is applied; and performing a process similar to the first trial coating on the coating surface of the second substrate, wherein if it is determined in the process of performing a process similar to the first trial coating using the second substrate that it is necessary to change the first depression amount, a process similar to the second trial coating is further performed on the second substrate.

[0155] (Appendix 15) The method for producing cell tissue described in Appendix 14 further comprises a step of performing a process similar to the first trial supply on the coating surface of the second substrate, and if it is determined that the first condition needs to be changed in the step of performing a process similar to the first trial supply using the second substrate, a process similar to the second trial supply is further performed on the second substrate.

[0156] 8 Plate, 8A Plate body, 8B Surface, 9A Well, 9A1 Set well position, 9A2 Actual measured well position, 20 Coating needle holder, 21 Coating material container, 24, 24A, 24B Coating needle, 35 Movable base, 41 Servo motor, 43 Cam, 44 Bearing, 45 Cam connecting plate, 46 Movable part, 81 Set position, 82 Actual measured position, 91 Wall surface, 92 Bottom surface, 100 Coating device, 101 X-axis stage, 102 Y-axis stage, 104 Needle coating mechanism, 105 Dropping mechanism, 106 Observation optical system, 107 Coating mechanism, A First coating liquid, B Second coating liquid, M Culture medium, P Lowest point.

Claims

1. A method for producing cell tissue, comprising: placing a first substrate for forming cell tissue within equipment; measuring the depth of the coating surface of the first substrate relative to the surface of the first substrate; and performing a first trial application of a first coating liquid to at least one coating section that is a part of the coating surface of the first substrate prior to supplying cell tissue to the first substrate, wherein the first trial application step includes applying the first coating liquid to one coating section with a first depression amount of an application needle that corresponds to the depth; and a first measurement step of measuring the diameter of the first coating liquid.

2. The method for producing cell tissue as described in claim 1, wherein the first trial application step further includes a first determination step of calculating a first difference between the diameter obtained in the first measurement step and a target diameter that is a target value for the diameter, and determining whether or not it is necessary to change the first pushing amount based on the first difference, and if it is determined in the first determination step that it is necessary to change the first pushing amount, a second trial application step of applying the first application liquid to one application portion with a second pushing amount that is different from the first pushing amount is further performed after the first trial application step.

3. The method for producing cell tissue according to claim 2, wherein the second trial application step includes: a step of reapplying the first application liquid to one application portion using a different pushing amount from the pushing amount used in the previous application, depending on the difference between the diameter in the previous application and the target diameter; a second measurement step of measuring the diameter of the first application liquid applied in the step of reapplying the first application liquid; and a determination step of determining whether the diameter of the first application liquid obtained in the second measurement step is within the range of the target diameter, and wherein the second trial application step is repeated until the diameter of the first application liquid in the second measurement step falls within the range of the target diameter.

4. The method for producing cell tissue according to claim 1 or 2, wherein the first application liquid is composed mainly of cells, a gel raw material, and a solvent.

5. The method for producing cell tissue according to claim 1 or 2, wherein the step of measuring the depth is performed by either measurement using a laser sensor or measurement using the autofocus of a camera.

6. The method for producing cell tissue according to claim 1 or 2, wherein the first substrate is a multi-well plate having a plurality of wells, and the application surface is the bottom surface of each of the plurality of wells.

7. The method for producing cell tissue described in claim 6, further comprising the steps of: measuring the depth of at least one of the plurality of wells in the depth measuring step; estimating, by calculation, the depth of the coating surface of other wells included in the first substrate other than the at least one well measured in the depth measuring step, relative to the surface of the first substrate; and determining the amount of depression for the other wells based on the depth of the other wells estimated in the estimation step.

8. A method for producing cell tissue as described in claim 7, wherein the method has at least two wells whose depths are measured among the plurality of wells, the other well is located at a position between positions where at least two of the wells whose depths are measured are located, and in the estimating step, the depth of the other well is calculated from the depths of the two wells measured in the depth measuring step based on a division ratio which is the ratio of the distance between each of the two wells whose depths are measured and the other well whose depth is to be estimated, and in the calculation, an estimated value of the depth is obtained under the assumption that the depth changes at the same rate between the two wells.

9. The method for producing cell tissue according to claim 1 or 2, further comprising a step of storing the depth measured in the step of measuring the depth.

10. A method for producing cell tissue as described in claim 1 or 2, comprising, prior to supplying cell tissue to the first substrate, a step of performing a first trial supply of the second application liquid to at least one application portion of the first substrate under first conditions of a discharge position, a discharge time, and a discharge pressure of the second application liquid, wherein the first trial supply step includes: a step of supplying the second application liquid to one application portion; and a second determination step of calculating a second difference between an output value under the first condition in the step of supplying the second application liquid and a target output which is a target value for the first condition, and determining whether or not the first condition needs to be changed in accordance with the second difference, and when it is determined in the second determination step that the first condition needs to be changed, a step of second trial supplying the second application liquid to one application portion under second conditions different from the first condition is further performed.

11. The method for producing cell tissue described in claim 10, wherein the second trial supplying step includes: a step of re-supplying the second application liquid to one application unit under conditions different from the discharge position, discharge time and discharge pressure conditions used in the previous supply, depending on the difference between the target output and the conditions of the discharge position, discharge time and discharge pressure of the second application liquid at the previous supplying step; and a step of examining whether the output value under the other conditions in the step of re-supplying the second application liquid is within the range of the target output, and wherein the second trial supplying step is repeated until the output value under the other conditions in the step of examining is within the range of the target output.

12. The method for producing cell tissue described in claim 10, wherein the output value of the discharge time and the output value of the discharge pressure are confirmed by checking whether or not the second application liquid has not been supplied within the required operating time.

13. The method for producing cell tissue described in claim 10, wherein in the first trial supplying step and the second trial supplying step of the second coating liquid, the second coating liquid is supplied so as to cover the first coating liquid, and the second coating liquid is mainly composed of a thickener and a solvent.

14. A method for producing cell tissue as described in claim 10, further comprising the steps of: placing a second substrate different from the first substrate in equipment; measuring the height position of the coating surface of the second substrate onto which the first coating liquid is applied; and performing a process similar to the first trial coating on the coating surface of the second substrate, wherein if it is determined in the process of performing a process similar to the first trial coating using the second substrate that it is necessary to change the first depression amount, a process similar to the second trial coating is further performed on the second substrate.

15. A method for producing cell tissue as described in claim 14, further comprising a step of performing a process similar to the first trial supply on the coating surface of the second substrate, and if it is determined that the first condition needs to be changed in the step of performing a process similar to the first trial supply using the second substrate, a process similar to the second trial supply is further performed on the second substrate.

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