Method for producing cell tissue

Abstract

To provide a method for producing a cell tissue, capable of reducing variation in application amount among a plurality of application parts.SOLUTION: The depth of the coated surface of the first substrate for cellular tissue formation relative to the surface is measured. Prior to supply of the cellular tissue to the first substrate, a first trial application of a first application liquid is performed on at least one application part that is a part of an application surface of the first substrate (S3). In the step of performing the first trial application (S3), the first application liquid is applied to one application portion by the first pushing amount of the application needle in accordance with the depth (S3 - 1). A first measuring step (S3 - 3) of measuring the diameters of the first coating liquids is performed.SELECTED DRAWING: Figure 11

Description

[Technical Field]

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

[0002] Japanese Patent Publication 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. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Patent No. 6461260 Summary of the Invention [Problem to be solved by the invention]

[0004] Multiwell plates with multiple wells are used to create (manufacture) cell tissues. The height of the bottom of each well (well bottom) in a commercially available multiwell plate is not uniform; it varies. For example, the bottom height 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 solution applied to each well. Here, the coating amount refers to the amount of coating solution transferred from the coating needle to the well. This is because the difference in the bottom height of each well causes the amount of pressure the lowered coating mechanism exerts on the well bottom to vary from well to well.

[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 bottoms of each well and the 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. [Means for solving the problem]

[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. [Effects of the Invention]

[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 variations in the amount of application among multiple application units. [Brief explanation of the drawings]

[0009] [Figure 1] 1 is a schematic front view showing a coating device according to a first embodiment. [Figure 2] 2 is a schematic diagram showing a needle coating mechanism of the coating device shown in FIG. 1. FIG. [Figure 3] FIG. [Figure 4]FIG. 2 is a schematic cross-sectional view showing a first example of the shape of a well formed in a plate. [Figure 5] FIG. 10 is a schematic cross-sectional view showing a second example of the shape of a well formed in a plate. [Figure 6] 1 is a flowchart showing an outline of a method for producing cell tissue according to the present embodiment. [Figure 7] 10 is a flowchart of a step (S1) performed on a first substrate. [Figure 8] FIG. 10 is a schematic diagram for explaining alignment correction in the step (S1A-1). [Figure 9] 10 is a flowchart of a step (S1) performed on a second substrate. [Figure 10] 10 is a flowchart of a mounting confirmation step (S2). [Figure 11] 10 is a flowchart of trial application (S3) of the first application liquid. [Figure 12] FIG. 4 is a schematic diagram showing the XY coordinates of a trial-applied first application liquid and the XY coordinates of its target position. [Figure 13] FIG. 10 is a schematic diagram illustrating the relationship between an area where the first application liquid is to be trial-applied and an area where the first application liquid is actually applied, for explaining the first determination step (S3-4). [Figure 14] 10 is a flowchart of trial supply (S4) of the second application liquid. [Figure 15] FIG. 1 is a schematic diagram showing an example of selection of wells for measuring the depth of the bottom surface from among wells contained in a multiwell plate as a first substrate. [Figure 16] FIG. 10 is a schematic diagram illustrating a method for estimating the depth of the bottom of a well whose depth has not been measured. [Figure 17] FIG. 1 is a schematic diagram showing the arrangement of 96 wells contained in a plate. [Figure 18] 18 is a graph showing measurements of the bottom height for each well of the plate of FIG. 17. [Figure 19] FIG. 4 is a schematic diagram showing variations in the amount of a first application liquid attached to an application needle. [Figure 20]FIG. 2 is a schematic diagram showing a state before a first application liquid is applied in the embodiment. [Figure 21] FIG. 3 is a schematic diagram showing the composition of a first coating liquid in the embodiment. [Figure 22] FIG. 3 is a schematic diagram showing a step of applying a first application liquid in the embodiment. [Figure 23] FIG. 3 is a schematic diagram showing a state after a first coating liquid has been applied in the embodiment. [Figure 24] FIG. 4 is a schematic view showing a step of supplying a second coating liquid in the embodiment. [Figure 25] FIG. 25 is a schematic diagram showing the state inside the well after the step of FIG. 24 has been performed. [Figure 26] FIG. 1 is a schematic diagram showing a step of supplying a culture medium in an embodiment. [Figure 27] FIG. 2 is a schematic diagram showing the state of cells in a first application liquid before culture. [Figure 28] FIG. 2 is a schematic diagram showing the state of cells in a first application liquid after culture. [Figure 29] 1 is a phase contrast microscope image immediately after application of the coating liquid in Example 1. DETAILED DESCRIPTION OF THE INVENTION

[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, such as 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 wells 9A of 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 above-described method for producing cell tissue may further be as follows: A first difference between the diameter 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 indentation amount needs to be changed is determined based on the first difference (first determination step). If it is determined in the first determination that the first indentation 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 indentation amount different from the first indentation amount is further performed after the first trial application step.

[0012] (Embodiment 1) (Configuration of coating device) FIG. 1 is a schematic front view of a coating apparatus according to a first embodiment. For ease of explanation, the X, Y, and Z directions are introduced. Referring to FIG. 1, the coating apparatus 100 includes a needle coating mechanism 104 and a dripping mechanism 105 as a coating mechanism 107 capable of supplying the coating material to be coated. In this specification, "coating" includes both supplying the coating material using a coating needle (described later) and supplying the coating material by dripping. Therefore, the former supply using a coating needle is sometimes referred to as "needle coating" in this specification. The 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 spaced apart in the X direction. The distance between them in the X direction is constant and does not change.

[0013] The trial application, which is the subject of this embodiment, is performed on the first and second application liquids, which are bioinks, but not on the culture medium. For this reason, 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 using visible light. However, the observation of the plate 8 is not limited to visible light, and may also be performed using infrared light, X-rays, ultrasound, or the like. Depending on the material of the plate 8, the plate 8 may also be observed using magnetism. The plate 8 observed using means other than visible light does not need to be transparent or translucent and may be opaque.

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

[0018] A bearing 44 is arranged so as to contact the cam surface of this cam 43. A cam connecting plate 45 is connected to this bearing 44. The other end of the cam connecting plate 45, opposite to one end connected to the bearing 44, is fixed to a movable part 46. A movable base 35 serving as a base body is connected to this movable part 46. A coating needle holder 20 is installed on this movable base 35. The coating needle holder 20 includes a coating needle 24. The coating needle 24 is capable of applying a coating material to, for example, a well 9A of the plate 8. The coating needle 24 is arranged so as to protrude from the coating needle holder 20 on the underside of the coating needle holder 20 (the lower side opposite the side on which the servo motor 41 is located). A coating material container 21 is arranged below the coating needle holder 20. The coating needle 24 is held in an inserted state in the coating material container 21.

[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, which is in contact with the cam surface of the cam 43, in the Z-axis direction varies in accordance with the rotation of the rotation shaft of the servo motor 41. The movable part 46 and the movable base 35 then move in the Z-axis direction in response to the positional variation of the bearing 44 in the Z-axis direction, thereby changing the position of the coating needle 24 in the Z-axis direction. In other words, the coating needle 24 can be reciprocated in the Z-axis direction. As a result of this movement, when the coating needle 24 is positioned upward in the Z-axis direction, the tip of the coating needle 24 is immersed in the coating material container 21, which contains the liquid material. In this state, the coating needle 24 projects downward from a tip hole in the bottom of the coating material container 21, thereby performing a coating operation. With the liquid material attached to the tip of the coating needle 24, the tip of the coating needle 24 projects from the tip hole of the coating material container 21 and exits the coating material container 21. At this time, the liquid material is pulled upward by surface tension, and a substantially constant amount of liquid material is attached to the tip of application needle 24. The attached liquid material is transferred into well 9A of plate 8 in this manner, 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 inside 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 8 rows in the X direction and 12 columns in the Y direction in FIG. 3, for a total of 96 wells. The planar shape of wells 9A is arbitrary, for example, circular.

[0023] Fig. 4 is a schematic cross-sectional view showing a first example of the shape of wells formed in a plate. As shown in Fig. 4, a plurality of wells 9A formed on the surface of plate 8 (plate body 8A) may be formed to have wall surfaces 91 extending in a direction intersecting with surface 8B and bottom surfaces 92 extending along surface 8B. Bottom surfaces 92 are the flat surfaces of wells 9A located at a position farthest 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 that is removed by the well 9A in a plan view. This configuration is also acceptable. However, as another example, the wall surface 91 may be perpendicular to the surface 8B. In other words, 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 be continuous with 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 position where it is bent relative to the wall surface 91). However, from the viewpoint 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 cell tissue) FIG. 6 is a flowchart outlining a 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, for example, 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 will be described later, 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. It 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 described assuming that it is a plate without wells.

[0029] Next, the depth of the coating surface is measured (S1). That is, the depth of the coating surface of the first substrate placed in the equipment in step (S0) 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 plate 8. Here, the first meaning of "depth" refers to the position coordinate (height) of the bottom surface 92 (coating surface) of well 9A in the vertical direction (Z direction). In other words, the "depth" of the bottom surface of well 9A is the vertical coordinate of the bottom surface 92 (coating surface). For example, "depth" may be the Z coordinate (negative value) of the bottom surface 92 when the Z coordinate of the surface of the first substrate (surface 8B in FIG. 4) is set as the reference value zero. If the first substrate does not have a well 9A, "depth" may also be considered the Z 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] FIG. 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 (coating surface) to which the first coating liquid and the second coating liquid will 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, first, XY coordinate data of an arbitrary position on plate 8 (multiwell plate) in FIG. 3 is set in advance. XY coordinates refer to X and Y coordinates (coordinate position in a plan view). Next, plate 8 is placed on XY stage 101. The XY coordinates of the position of plate 8 that has actually been placed are measured.

[0033] FIG. 8 is a schematic diagram illustrating alignment correction in step (S1A-1). FIG. 8 shows a state in which plate 8 is viewed in a plane 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 so that its outline is located at set well position 9A1. As an example, FIG. 8 shows set well positions 9A1 for a pair of diagonally opposite corner wells. However, based on measurements in this step, plate 8 is actually positioned at actual measurement position 82. Based on the 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 height of the well bottom, is measured (S1A-2). That is, the coordinate of the vertical (Z-direction) height of the bottom 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 position in the Z direction 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 92 relative to the surface 8B (S1A-3). The height of the well bottom may also be measured by acquiring an image using a combination of lighting and an observation device such as a camera, and 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 flat and has a uniform height throughout, alignment correction of the second substrate is not necessary. With glass slides and 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 the coated surface is measured only for a portion (coated portion) of the coated surface of the well bottom or slide glass, etc.

[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 position of the well bottom is necessary to estimate the Z-direction position 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 position of the well bottom 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, attachment confirmation (S2) is performed in the coating mechanism. FIG. 10 is a flowchart of the attachment confirmation step (S2). As shown in FIG. 10, the needle coating mechanism 104 is attached to the coating device 100. The coating needle 24 and the coating material container 21 are attached to the needle coating mechanism 104 (S2-1). It is confirmed (S2-2) whether the coating needle 24 attached to the needle coating mechanism 104 moves normally in the vertical direction (Z direction). 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 attachment of the coating needle 24 is redone, and drive confirmation (S2-2) is performed again. Steps (S2-1) and (S2-2) are repeated until the drive state 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 the arrow GOOD. That is, the drip mechanism 105 is attached to the coating device 100 (S2-3). The discharge time and discharge 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 application liquid is performed (S3). This is a step in which, prior to the actual application for supplying cellular tissue to the first substrate, the first application liquid is trial-applied (trially applied) from the needle application mechanism 104 to the application portion, which is 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 (different from the first substrate to be actually coated) whose bottom surface 92 has its depth measured in step (S1). 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 on the coating surface (such as the bottom surface of a well or the surface of a slide glass) whose Z-direction position 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 formed in the plate 8 and whose Z-direction position has been measured, other than the well 9A to be used for the main coating. 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. A positional deviation (error) occurs 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] Referring again to Figure 11, next, 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 the amount of push-up needs to be changed (S3-4). Specifically, in the first determination step, it is determined whether or not the first amount of push-up used when applying the first application liquid in step (S3-1) needs to be changed. 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 that is a target value for that diameter, is calculated. Depending on the first difference, it is determined whether or not the first amount of push-up needs to be changed.

[0049] FIG. 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 the 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 amount of pushing in the next trial application is increased compared to the immediately preceding trial application. 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 amount of pushing in the next trial application is decreased compared to the immediately preceding trial application.

[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 centered around 1000 μm and has a range of ±10%. In other words, in this example, the target diameter is 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 process thereafter (step (S3-5) and thereafter, which will be described next) is 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 no further trial application is 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 application 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 process 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 application liquid is again applied by a push-up amount different from the immediately preceding application (S3-5). At this time, a second trial application of the first application 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] In the step (S3-5) of re-applying the first coating liquid, a second measurement step (S3-6) is performed to measure the diameter of the first coating liquid applied. 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 R2 (see FIG. 13) is within the range of the target diameter R1 in the determination step (S3-7) and another trial application is not required, 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 R2 shown in FIG. 13 is outside the range of the target diameter R1 in step (S3-7), it is determined that the second push-in amount needs to be changed and another trial application is required. 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) is within the range of the target diameter R1.

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

[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). In this way, when the second trial coating step is performed multiple times, the second push-in amount in each step is different from each other. In other words, multiple different amounts are assumed as the second push-in amount. 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 step was the first trial coating) is taken into consideration.

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

[0060] When the trial application (S3) of the first application liquid in FIG. 11 is performed on a second substrate different from the first substrate, the application surface (application portion) of the second substrate is subjected to the same process as the first trial application. It may be determined that the first depression amount needs to be changed in the process of performing the same process as the first trial application using the second substrate. In that case, the second substrate is further subjected to the same process as the second trial application. In this case, the first trial application and the second trial application are not necessarily performed on the bottom surface (application 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). In this way, the application amount of the first application liquid, etc., is adjusted.

[0061] 11, the process (S3) using the first substrate and the process (S3) using the second substrate are generally the same. 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." 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). In other words, 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 drip 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 the 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 by measuring, for example, the Z coordinate of the tip of the drip mechanism 105. The output value of the discharge time and the output value of the discharge pressure are confirmed by checking whether or not any of the second coating liquid has not been supplied within the required operation time. This results in the output value of the discharge time and the output value of 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. By optimizing the lens magnification of the observation optical system, it is possible to measure the diameter of the second coating liquid. 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 is within the target value range in step (S4-4) 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 is outside the target value range in step (S4-4), it is determined in the second determination step that the first condition needs to be changed and another trial supply is required. 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, the second coating liquid is supplied to the application portion of one well 9A under second conditions, such as discharge conditions different from the first conditions, taking into account the second difference. In the second trial supply, the second coating liquid is preferably 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 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 application 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 application 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 discharge position of the second application liquid) obtained in the examination step (S4-7) is within the target output range.

[0074] When the second trial supply process 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). In this way, when the second trial supply process is performed multiple times, the second conditions are different for each process. 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 process and the target value of that condition (the second difference if the immediately preceding process was the first trial supply) is taken into account.

[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) during 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 in 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 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. In this way, the amount of the second coating liquid dispensed, etc., is adjusted.

[0077] 14, the process (S4) using the first substrate and the process (S4) using the second substrate are generally the same. 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 (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 main application are completed, the main 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 processing 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 multi-well 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 of 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] FIG. 15 is a schematic diagram showing an example of well selection for measuring bottom depth from among wells contained in a multiwell plate serving as a first substrate. As shown in FIG. 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. FIG. 15 shows a total of 12 types of well selection modes (1) to (12) for measuring bottom depth (position in the Z direction). Wells selected for measuring bottom depth are hatched. Hereinafter, for example, a well at the intersection of row A and column 1 may be referred to 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, it may be a single well selected arbitrarily, such as A1 in (1). It may be wells at the two outermost diagonally opposite corners, such as A1 and H12 in (2). It may be wells at the four outermost corners, such as A1, H1, A12, and H12 in (3). It may be a total of nine wells, including the four outermost corners, the center (D6), and the centers of the four outermost sides, as in (4). These nine wells are arranged at approximately equal intervals. All wells in column 1 may be selected, as in (5). All wells in row A may be selected, as in (6). All wells in both column 1 and row A may be selected, as in (7). All wells in columns 1, 12, row A, and row H, covering the entire circumference, may be selected, as in (8). All wells in rows A, C, E, and G may be selected, as in (9). 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 Figure 16, well 2, whose depth has not been measured, is located in a position in the Y direction between the positions of wells 1 and 3, whose depths have been measured. 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 considered to be 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 Figure 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 by the vertical axis (well bottom height) in Fig. 16. Well 2 is located midway between Well 1 and Well 3 in the Y direction. That is, Well 2 bisects Wells 1 and 3 in a 1:1 ratio. The bottom height of the well at any position between Well 1 and Well 3 is obtained from the height direction position of the straight line connecting Well 1 and Well 3 (at the center). Since the bottom height (depth) changes at the same rate between Well 1 and Well 3, the slope of the straight 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 straight line connecting Well 1 and Well 3 in Fig. 16. Similarly, the bottom height of Well 4 can be predicted from the slope of the straight line connecting Well 3 and Well 5. For example, if Well 4 exists at a position that bisects Wells 3 and 5 in a 1:2 ratio and the bottom height of Well 5 is 60 μm, the bottom height of Well 4 is 20 μm.

[0087] Expressing the above in a mathematical formula gives the following formula (1). Let h(r) be the bottom height of the well r whose bottom height is to be estimated in Fig. 16. Let h(m) be the measured bottom height of Well m, and h(n) be the measured bottom height of Well n different from Well n. Wells m, r, and n are arranged so as to lie on a straight line in rows A to H or columns 1 to 12 of Fig. 15. The magnitude relationship is m < r < n. For example, in Fig. 16, Wells m, r, and n are Wells 1, 2, 3 or Wells 3, 4, 5. At this time, the value h(r) to be obtained is

[0088]

Number

[0089] …(1) obtained as follows. h(n) in the second term of the above formula (1) may be h(m).

[0090] Alternatively, the depth of the coating surface of unmeasured wells may be estimated by calculation using 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, and is basically configured similarly to FIG. 15. Therefore, 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).

[0091] 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 case where a plate manufactured by a manufacturer showing the data in FIG. 18(A) is used. First, data showing the bottom depths of 96 wells, as shown in FIG. 18(A), is stored in the storage medium of the coating device 100. In the data in FIG. 18(A), for example, the well bottoms in columns 1 and 12 tend to be high, and the well bottoms in column 6 tend to be low. Furthermore, particularly in columns 6 to 12, the well bottoms in rows A, B, and C tend to be high.

[0092] 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 height of each well other than well A1 increases or decreases in the same manner as the stored data, the height of each well other than well A1 is estimated.

[0093] For example, consider a case where the measured bottom height of well A1 on a plate to be measured is 10 μm. In FIG. 18(A), the bottom height of well A1 is 0 μm. Therefore, the bottom position of the well on the plate to be measured is considered to be 10 μm higher than the data in FIG. 18(A). Therefore, for example, the bottom height of well A7 on that plate is estimated to be -90 μm, 10 μm higher than the -100 μm shown in FIG. 18(A).

[0094] Furthermore, the depth of the coating surface of unmeasured wells may be estimated by the following third method: In the third method, the depth of the bottom surface may be estimated by calculation using artificial intelligence.

[0095] The bottom height (depth) of the wells 2, 4 is found (estimated) by any of the above-described first to third methods. Based on the estimated value, the pushing amount of the application needle 24 into the wells 2, 4 is determined. The pushing amount here is the pushing amount of the application needle 24 in the trial application (S3) of the first application liquid in the first embodiment, etc.

[0096] (Action and effect) The following will explain the effects of the first and second embodiments together, while also touching upon the background art and problems that led to the present embodiments.

[0097] 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 part 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.

[0098] As shown in FIG. 18, the bottom heights of multiple wells 9A formed in one plate 8 vary. The trends are different between the upper and lower graphs in FIG. 18. In other words, the tendency of variation in the bottom heights of the wells 9A is not uniquely determined and may vary depending on the 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 heights of the wells of the plate 8 to be determined in advance. This makes it possible to adjust the depth of the coating needle 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 FIG. 13 from being mixed within a single plate 8.

[0099] However, simply determining 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 height 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 application needle 24A and application needle 24B are pressed in by the same amount, application needle 24A will apply a larger amount of first application liquid A. In other words, even if the bottom height of the well is measured and the application needle is pressed in based on that result, a difference in the amount applied will occur, as shown by A and B in Figure 13.

[0100] Therefore, in this embodiment, a first trial application of the first application liquid is performed in advance. The first application liquid is applied with a first push-in amount corresponding to the measured value of the depth of the application surface of well 9A, etc., and the diameter of the applied first application liquid is measured. This suppresses environmental variations that inevitably occur between work days, and makes it possible to determine in advance with high precision the amount the application needle should be pushed in for each well bottom height.

[0101] The first coating liquid A is applied to the coating needle 24 each time the coating needle 24 is pressed into the substrate to apply the liquid. In other words, the number of times the first coating liquid A is applied to the coating needle 24 is equal to the number of times the coating needle 24 is pressed into the substrate to apply the liquid. Variation in the amount of the first coating liquid A applied to 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 amount of coating during the actual coating operation can be understood immediately before the actual coating operation, variation in the amount of coating between the coating portions, such as the multiple wells 9A formed on the plate 8, during the actual coating can be reduced. As a result, the coating liquid can be supplied stably during the actual coating operation on that day.

[0102] 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 pushing amount needs to be changed is determined based on the first difference. If it is determined in the first determination step that the first pushing amount needs to be changed, a second trial application step of applying the first application liquid to one application area with a second pushing amount different from the first pushing amount is further performed after the first trial application step.

[0103] Based on the diameter of the first application liquid obtained in the first measurement step, the push-in amount is adjusted as needed by the second trial application, thereby reducing environmental variations between different work days and further enhancing the effectiveness of predetermining the push-in amount of the application needle for each well bottom height.

[0104] 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. This allows the optimal pressing amount to 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 plate 8.

[0105] In the above-described method for producing cell tissue, the first substrate is 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.

[0106] In the method for producing the cell tissue, the depth of at least one well 9A among the multiple wells is measured in the depth measuring step. The depth of the coating surface (bottom surface 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 is estimated by calculation. The amount of depression for the other wells is determined based on the depth of the other wells 9A estimated in the depth estimating step.

[0107] 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 depth as other adjacent wells 9A without relying on calculation, for example, 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, for example, measuring the depth of all wells 9A has the advantage of being able to determine the bottom height of the well 9A with high precision. On the other hand, measuring all wells 9A has the disadvantage of increasing 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 allowing the depth of more (all) wells 9A to be determined with high precision.

[0108] In the method for producing the cell tissue, at least two wells 9A are provided among the plurality of wells, the depths of which are to be measured. The other wells 9A are located between the positions of at least two of the plurality of wells 9A, the depths of which are to be measured. In the depth estimation step, the depths of the other wells 9A are 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 distances between each of the two wells 9A whose depths are to be measured and the other well 9A whose depth is to be estimated. In the calculation, a depth estimate is obtained under the assumption that the depths of the two wells 9A change at the same rate. In this way, the depths of more (all) wells 9A can be determined with high accuracy while reducing the operation time.

[0109] The above-described method for producing cell tissue preferably further comprises a step of storing the measured depth in the depth measurement step. This allows the depth 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 amount of indentation can be controlled with high precision using the highly accurately measured and stored values. This effectively reduces variation in the amount of coating.

[0110] 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, including 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 step of supplying the second coating liquid 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.

[0111] In the trial supply of the second coating liquid, the process of controlling the output value is performed using essentially the same procedure as in the trial supply of the first coating liquid. This provides the same effects as in 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.

[0112] When dispensing the second coating liquid, the height position of the coating surface, such as the bottom of the well, has less of an effect on the dispensing state compared to when the coating needle is pressed in to dispense the first coating liquid. However, adjusting the Z-direction discharge position of the second coating liquid is important for controlling the dispensing state of the second coating liquid. For this reason, the discharge position is adjusted. Dispensing time and pressure are also important for controlling the dispensing state of the second coating liquid, so they are adjusted.

[0113] In the above-described method for producing cell tissue, the second trial supply step resupplies 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 of the conditions for the discharge position, discharge time, and discharge pressure of the second application liquid in the previous supply. It is examined whether the output value of the other conditions in the step of resupplying the second application liquid is within the target value range. The second trial supply step is repeated until the output value of the other conditions in the examination step falls within the target value range. In this way, variation in the amount of second application liquid supplied can be reduced.

[0114] 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 step 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.

[0115] 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).

[0116] 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 accuracy, taking into account the daily variation in the amount of adhesion to the coating needle 24 on a given day.

[0117] 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 through trial coating using the second substrate, this relationship can also be used during the actual coating using the first substrate.

[0118] 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 checked, the same effect as when the first substrate is used can be obtained.

[0119] (Explanation of the specific manufacturing method using diagrams) Here, an example using a multiwell plate is illustrated. However, similar procedures apply when using equipment other than a multiwell plate, such as a dish or a glass slide. FIG. 20 is a schematic diagram showing the state before the first coating liquid is applied in an embodiment. As shown in FIG. 20, the tip of the applicator needle 24 constituting the bioprinter is first immersed in the first coating liquid A (first bioink), and the first coating liquid A is attached to the tip of the applicator needle 24. FIG. 21 is a schematic diagram showing the composition of the first coating liquid in an embodiment. As shown in FIG. 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 FIG. 21 include C1 and C2, which will be described later. FIG. 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 applicator needle 24 to which the first coating liquid A is attached comes into contact with, for example, the bottom surface 92 (see FIG. 4) of the well 9A (see FIG. 4). This is achieved by moving the application needle 24, to which the first application liquid A has been attached, downward as shown by arrow M1 in FIG. 20. As a result, the first application liquid A is applied to the bottom surface 92 of the well 9A. FIG. 23 is a schematic diagram showing the state after the first application liquid has been applied in this embodiment. As shown in FIG. 23, the application needle 24 then moves upward as shown by arrow M2. In this way, the first application liquid A is applied to a container such as the well 9A using a bioprinter of the so-called pin type (a needle type such as the needle application mechanism 104 in FIG. 1).

[0120] 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 the 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 the second coating liquid B is not limited to this. The second coating liquid B may be supplied by any method selected from the group consisting of a pin method, an inkjet method, a dispenser method (a method using the dropping mechanism 105 of Fig. 1), and manual supply using a pipette.

[0121] 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.

[0122] 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.

[0123] (material) The first coating liquid A is composed of cells to be cultured, a gel (a gel raw material if liquid) for producing (manufacturing) cell tissue, and a first solvent. In addition to the above, various additives may be added to the first coating liquid A.

[0124] The type of cell is not particularly limited. Normal cells or cells derived from various diseases may be used. Alternatively, cells that have undergone gene transfer, genetic modification, or genetic recombination may be used. Cells may be derived from any animal (animal origin) including humans, mice, rats, and monkeys. Cells may be differentiated cells derived from stem cells. In other words, cells may be differentiated cells derived from iPS cells or ES cells. Cells may be mesenchymal stem cells. Cells may be primary cells or established cell lines.

[0125] The cell types may be any of neurons, cardiomyocytes, fibroblasts, vascular endothelial cells, hepatocytes, Kupffer cells, hepatic stellate cells, pit cells, epithelial cells, and skeletal muscle cells, i.e., cells derived from various organs. Neurons include central neurons, sympathetic neurons, parasympathetic neurons, sensory neurons, interneurons, motor neurons, microglia, astrocytes, oligodendrocytes, ependymal cells, Schwann cells, and satellite cells. Cardiomyocytes include ventricular myocytes and atrial myocytes. First coating solution A contains one or more of the above cell types. In other words, first coating solution A may contain multiple cell types.

[0126] The density of the cells in the first coating liquid A is not particularly limited. However, the density of the cells is, for example, 1×10 2 cells / mL or more 1×10 9 It may be less than cells / mL.

[0127] First Coating Liquid A does not necessarily have to contain a gel (a gel raw material before solidification: hereinafter referred to as a gel). For example, First Coating Liquid A may contain either a natural polymer or a synthetic polymer. When a gel is used in First Coating Liquid A, the material of the gel is not particularly limited. The gel in First Coating Liquid A may be, for example, any of collagen, fibrin, Matrigel, gelatin, sodium alginate, gelatin methacryloyl (GelMA), cellulose, cellulose nanofiber, chitin, chitosan, chitin nanofiber, chitosan nanofiber methylcellulose, carboxymethylcellulose, hydroxybutylcellulose, sodium alginate, sodium hyaluronate, polyethylene glycol, gellan gum, carrageenan, pectin, xanthan gum, gelatin, agarose, and polyvinyl alcohol.

[0128] The material of the first solvent is not particularly limited. However, the first solvent may be the same material as the culture medium or a buffer solution. For example, the first solvent may be any of DMEM, DMEM / Ham F-12, αMEM, RPMI-1640, Williams' medium, M199, commercially available culture media for various cells, PBS solution (+ or -), Tris buffer, and Tyrode's buffer.

[0129] The materials of the various additives in the first coating liquid are not particularly limited. The various additives may be drugs acting on cells, cell growth factors, cytokines, hormones, transcription factors, ECM, proteins, antibodies, thickeners, salts, etc. The various additives may be low-molecular-weight compounds, medium-molecular-weight compounds, or high-molecular-weight compounds. For example, the additives include T3, T4, IGF (insulin-like growth factor: IGF-I), epidermal growth factor (EGF), TGF, basic fibroblast growth factor (bFGF), fibroblast growth factor (FGF2), vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), granulocyte-colony stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), platelet-derived growth factor (PDGF), EPO, TPO, hepatocyte growth factor (HGF), insulin, dexamethasone, isoproterenol, B27 (registered trademark) supplement, N2 supplement, fetal bovine serum (FBS), collagen, fibrin, Matrigel, gelatin, fibronectin, vitronectin, laminin, proteoglycan, nidogen, ROCK The inhibitor may be any of sodium alginate, gelatin methacryloyl (GelMA), cellulose, cellulose nanofiber, chitin, chitosan, chitin nanofiber, chitosan nanofiber, methylcellulose, carboxymethylcellulose, hydroxybutylcellulose, sodium alginate, sodium hyaluronate, polyethylene glycol, gellan gum, carrageenan, pectin, xanthan gum, gelatin, agarose, and polyvinyl alcohol.

[0130] 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.

[0131] 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.

[0132] The medium for culturing cells, i.e., medium M (see FIG. 24) 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. Various additives may also be added to medium M. The materials of the various additives to medium M may be the same as those of the various additives to first coating liquid A or second coating liquid B described above.

[0133] The first substrate for culturing cells is, for example, a plate 8 having wells 9A. The plate 8 is not limited to having 96 wells 9A as 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 multiwell 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 desk 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.

[0134] The surface of the first substrate may be cell-adhesive, and the cell-adhesive first substrate is 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. [Example]

[0135] An experiment was conducted to actually form cell tissue based on the cell tissue manufacturing method 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.

[0136] The plate 8 used was a multi-well plate having 96 wells 9A as shown in Figure 3. Each droplet described below was supplied into the wells 9A.

[0137] 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 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% FBS by mass.

[0138] 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 wells to cover them. Medium M was dripped 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 the specific manufacturing method using diagrams).

[0139] 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 for applying the first coating liquid was corrected as follows. The difference between the first diameter measurement (see S3-3 in Figure 11) and the first diameter measurement (see S3-1 in Figure 11) using the first depression amount 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.

[0140] 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 a reasonable diameter was stably formed.

[0141] (Addendum) Various aspects of the present disclosure are summarized below as appendices.

[0142] (Appendix 1) placing a first substrate for cell tissue formation in a facility; 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 application liquid to at least one application portion that is a part of the application surface of the first substrate prior to supplying the cell tissue to the first substrate, The first trial application step includes: applying the first application liquid to one of the application portions with a first pushing amount of an application needle corresponding to the depth; a first measuring step of measuring the diameter of the first application liquid.

[0143] (Appendix 2) 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 push-in amount based on the first difference; The method for producing cell tissue described in Appendix 1, wherein if it is determined in the first judgment step that the first depression amount needs to be changed, after the first trial application step, a second trial application step of the first application liquid to one application portion at a second depression amount different from the first depression amount is further performed.

[0144] (Appendix 3) The second trial application step includes: a step of applying the first application liquid again to one of the application parts by a push-in amount different from the push-in amount used in the previous application, in accordance with a difference between the diameter at the previous application and the target diameter; a second measuring step of measuring a diameter of the first coating liquid applied in the step of applying the first coating liquid again; a determination step of determining whether the diameter of the first coating liquid obtained in the second measurement step is within the target diameter range, The method for producing cell tissue described in Appendix 2, 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.

[0145] (Appendix 4) 4. The method for producing a cell tissue according to any one of claims 1 to 3, wherein the first application liquid contains cells, a gel raw material, and a solvent as main components.

[0146] (Appendix 5) 5. The method for producing cell tissue according to any one of appendices 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.

[0147] (Appendix 6) the first substrate is a multiwell plate having a plurality of wells; The method for producing a cell tissue according to any one of appendixes 1 to 5, wherein the application surface is the bottom surface of each of the plurality of wells.

[0148] (Appendix 7) In the step of measuring the depth, the depth of at least one of the plurality of wells is measured; a step of 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 step of measuring the depth, relative to the surface of the first substrate; The method for producing cell tissue described in Appendix 6 further comprises a step of determining the amount of depression into the other well based on the depth of the other well estimated in the estimation step.

[0149] (Appendix 8) The depth measurement device includes at least two wells among the plurality of wells, the other well is disposed at a position between positions where at least two of the plurality of wells are disposed at the wells for which the depth is to be measured, In the estimation 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 a ratio of the distance between each of the two wells whose depths are measured and the other well whose depth is to be estimated, A method for producing cell tissue described in Appendix 7, wherein the calculation estimates the depth under the assumption that the depth changes at the same rate between the two wells.

[0150] (Appendix 9) The method for producing a cell tissue according to any one of appendices 1 to 8, further comprising the step of storing the depth measured in the step of measuring the depth.

[0151] (Appendix 10) a step of first trial supplying 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, prior to supplying the cellular tissue to the first substrate; The first trial supplying step includes: supplying the second coating liquid to one of the coating units; a second determination step of calculating a second difference between an output value under the first condition in the step of supplying the second coating liquid and a target output that is a target value under the first condition, and determining whether or not it is necessary to change the first condition based on the second difference; A method for producing cell tissue described in any one of Appendices 1 to 9, wherein if it is determined in the second judgment step that the first conditions need to be changed, a step of second trial supplying the second application liquid to one application section under second conditions different from the first conditions is further performed.

[0152] (Appendix 11) The second trial supplying step includes: supplying the second application liquid again to one of the application units under conditions different from the conditions of the discharge position, the discharge time, and the discharge pressure used during the immediately previous supply, in accordance with a difference between the target output and the conditions of the discharge position, the discharge time, and the discharge pressure of the second application liquid during the immediately previous supply; and examining whether or not the output value under the other conditions in the step of supplying the second coating liquid again is within the range of the target output, A method for producing cellular tissue as described in Appendix 10, wherein the second trial supply step is repeated until the output value of the other condition in the examination step falls within the range of the target output.

[0153] (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.

[0154] (Appendix 13) 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, 13. The method for producing a cell tissue according to any one of claims 10 to 12, wherein the second application liquid contains a thickener and a solvent as main components.

[0155] (Appendix 14) placing a second substrate different from the first substrate in the equipment; measuring a position in a height direction of the coating surface of the second substrate onto which the first coating liquid is coated; and performing a process similar to the first trial coating on the coating surface of the second substrate, A method for producing cell tissue described in any one of Appendices 10 to 13, wherein if it is determined that the first pushing amount needs to be changed in the process of performing a process similar to the first trial application using the second substrate, a process similar to the second trial application is further performed on the second substrate.

[0156] (Appendix 15) and performing a process similar to the first trial supply on the coating surface of the second substrate, A method for producing cell tissue as described in Appendix 14, wherein if it is determined that the first conditions need to be changed in the process 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. [Explanation of symbols]

[0157] 8 plate, 8A plate body, 8B surface, 9A well, 9A1 set well position, 9A2 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 measured position, 91 wall surface, 92 bottom surface, 100 coating device, 101 X-axis stage, 102 Y-axis stage, 104 needle coating mechanism, 105 dripping mechanism, 106 observation optical system, 107 coating mechanism, A first coating liquid, B second coating liquid, M culture medium, P lowest point.

Claims

1. placing a first substrate for cell tissue formation in a facility; 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 application liquid to at least one application portion that is a part of the application surface of the first substrate prior to supplying the cell tissue to the first substrate, The first trial application step includes: applying the first coating liquid to one of the coating portions with a first pushing amount of a coating needle corresponding to the depth; a first measuring step of measuring the diameter of the first coating liquid.

2. 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 push-in amount based on the first difference; 2. The method for producing cell tissue according to claim 1, wherein, if it is determined in the first judgment step that the first pushing amount needs to be changed, after the first trial application step, a second trial application step of the first application liquid to one application portion at a second pushing amount different from the first pushing amount is further performed.

3. The second trial application step includes: a step of applying the first application liquid again to one of the application parts by a push-in amount different from the push-in amount used in the previous application, in accordance with a difference between the diameter at the previous application and the target diameter; a second measuring step of measuring a diameter of the first coating liquid applied in the step of applying the first coating liquid again; a determination step of determining whether the diameter of the first coating liquid obtained in the second measurement step is within the target diameter range, The method for producing cell tissue according to claim 2 , 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 contains cells, a gel raw material, and a solvent as main components.

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 autofocus of a camera.

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

7. In the step of measuring the depth, the depth of at least one of the plurality of wells is measured; a step of 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 step of measuring the depth, relative to the surface of the first substrate; The method for producing cell tissue according to claim 6 , further comprising the step of determining a pressing amount for the other well based on the depth of the other well estimated in the estimation step.

8. The depth measurement device includes at least two wells among the plurality of wells, the other well is disposed at a position between positions at which at least two of the plurality of wells are disposed of the wells whose depths are to be measured; In the estimation 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 a ratio of the distance between each of the two wells whose depths are measured and the other well whose depth is to be estimated, The method for producing a cell tissue according to claim 7 , wherein the depth is estimated under the assumption that the depth changes at the same rate between the two wells in the calculation.

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

10. prior to supplying the cellular tissue to the first substrate, performing a first trial supply of the second coating liquid to at least one coating portion of the first substrate under first conditions of a discharging position, a discharging time, and a discharging pressure of the second coating liquid; The first trial supplying step includes: supplying the second coating liquid to one of the coating units; 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 under the first condition, and determining whether or not it is necessary to change the first condition based on the second difference, The method for producing cell tissue described in claim 1 or 2, wherein if it is determined in the second judgment process that the first conditions need to be changed, a second trial supply process of the second application liquid to one application section under second conditions different from the first conditions is further performed.

11. 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 conditions of the discharge position, the discharge time, and the discharge pressure used during the immediately previous supply, in accordance with a difference between the target output and the conditions of the discharge position, the discharge time, and the discharge pressure of the second application liquid during the immediately previous supply; a step of examining whether or not the output value under the other conditions in the step of supplying the second coating liquid again is within the range of the target output, The method for producing a cellular tissue according to claim 10 , wherein the second trial supplying step is repeated until the output value of the other condition in the examining step falls within the range of the target output.

12. The method for producing cell tissue according to 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. 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, The method for producing cell tissue according to claim 10 , wherein the second coating liquid contains a thickener and a solvent as main components.

14. placing a second substrate different from the first substrate in a facility; measuring a position in a height direction of the coating surface of the second substrate onto which the first coating liquid is coated; and performing a process similar to the first trial coating on the coating surface of the second substrate, The method for producing cell tissue described in claim 10, wherein if it is determined that the first pushing amount needs to be changed in a process of performing a process similar to the first trial application using the second substrate, a process similar to the second trial application is further performed on the second substrate.

15. and performing a process similar to the first trial supply on the coating surface of the second substrate, The method for producing cell tissue described in claim 14, wherein if it is determined that the first conditions need to be changed in a process 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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