Method for manufacturing cell tissue structures

Abstract

This invention provides a method for producing cell tissue structures that are highly stable and reproducible. [Solution] The number of cells in the cell suspension and the cell diameter are counted (S10). The cell volume is calculated from the number of cells and cell diameter obtained in the counting step (S20). A cell pellet is obtained by centrifuging the cell suspension containing the cells whose cell volume has been calculated (S30). The cell pellet volume, which is the total volume of the cell pellet, is calculated (S40). Based on the cell pellet volume, a solvent containing biomaterials is added to the cell pellet so that the cells have a desired cell volume fraction, and a mixture is obtained (S50).

Description

[Technical Field]

[0001] This invention relates to a method for producing cell tissue structures. [Background technology]

[0002] International Publication No. 2019 / 088224 (Patent Document 1) describes a cell suspension filled in a coating solution container having a through-hole at the bottom. Patent Document 1 discloses a method of applying the cell suspension as a coating solution to an object using a coating needle. In Patent Document 1, the coating solution is attached to the tip of the coating needle, and the coating solution is brought into contact with the object to be coated or with a coating solution that has already been applied. This creates a cell tissue structure. [Prior art documents] [Patent Documents]

[0003] [Patent Document 1] International Publication No. 2019 / 088224 [Overview of the Initiative] [Problems that the invention aims to solve]

[0004] In Patent Document 1, variations occur in the amount of liquid component in the coating solution applied to the substrate and the cell number concentration therein, depending on the diameter of the cells contained in the coating solution and the properties of the solvent added thereto. For this reason, it is difficult to produce a cell tissue structure with high stability and reproducibility using Patent Document 1. "Solvent" refers to gelling agents or gelling initiators (biomaterials), and thickeners, etc. "Cell number concentration" is an index that shows the number of cells contained per unit volume of the coating solution as a concentration. The cell number concentration is calculated from the number of cells in the coating solution.

[0005] The present invention has been made in view of the above-mentioned problems. The object of the present invention is to provide a method for producing cell tissue structures with high stability and reproducibility. [Means for solving the problem]

[0006] In the method for manufacturing a cell tissue structure according to the present disclosure, the number of cells, which is the number of cells contained in the cell suspension, and the cell diameter are counted. The cell volume is calculated from the number of cells and the cell diameter obtained in the counting step. A cell pellet is obtained by centrifuging the cell suspension containing the cells for which the cell volume has been calculated. The cell pellet volume, which is the total volume of the cell pellet, is calculated. Based on the cell pellet volume, a solvent containing a biological material is added to the cell pellet so that the cells have a desired cell volume fraction, and a mixed solution is obtained.

Advantages of the Invention

[0007] According to the present disclosure, the amount of the solvent added to the cell suspension is adjusted so as to achieve a predetermined cell volume fraction. Therefore, it is possible to provide a method for manufacturing a cell tissue structure with high stability and reproducibility.

Brief Description of the Drawings

[0008] [Figure 1] It is a schematic diagram of a coating device according to the present embodiment. [Figure 2] It is a schematic diagram showing the coating mechanism of the coating device shown in FIG. 1. [Figure 3] It is a schematic diagram showing the coating process by the coating needle. [Figure 4] It is a schematic perspective view of a well plate. [Figure 5] It is a flowchart showing the method for manufacturing a cell tissue structure according to the present embodiment. [Figure 6] It is a chart listing the processes performed in step (S10) of FIG. 5. [Figure 7] It is a schematic diagram showing the state before the first coating solution is applied in the embodiment. [Figure 8] It is a schematic diagram showing the composition of the first coating solution in the embodiment. [Figure 9] It is a schematic diagram showing the step of applying the first coating solution in the embodiment. [Figure 10] It is a schematic diagram showing the state after the first coating solution is applied in the embodiment. [Figure 11] This is a schematic diagram showing the process of supplying the second coating solution in the embodiment. [Figure 12] This is a schematic diagram showing the state of the well after the process shown in Figure 11 has been completed. [Figure 13] This is a schematic diagram showing the process of supplying the culture medium in the embodiment. [Figure 14] This is a schematic diagram showing the state of cells in the first coating solution before culture. [Figure 15] This is a schematic diagram showing the state of cells in the first coating solution after culture. [Figure 16] This is a photograph of the cell tissue structure prepared in Example 1. [Figure 17] This is a photograph showing the phase-contrast observation results of the cell tissue structure prepared in Example 3. [Figure 18] This is the result of phase-contrast observation of a cellular tissue structure of 50 cardiomyocytes prepared on a single 96-well plate. [Figure 19] This is a photograph showing the phase-contrast observation results of the cell tissue structure prepared in Example 4. [Figure 20] This is a photograph showing the phase-contrast observation results of the cell tissue structure prepared in Example 5. [Modes for carrying out the invention]

[0009] This embodiment will be described below with reference to the drawings. (Device configuration) Figure 1 is a schematic diagram of a coating apparatus according to this embodiment. The coating apparatus according to this embodiment will be explained using Figure 1. For the sake of explanation, the X, Y, and Z directions are introduced. Referring to Figure 1, the coating apparatus according to this embodiment mainly comprises a processing chamber, a Y-axis table 2, an X-axis table 1, a Z-axis table 3, a coating mechanism 4, an observation optical system 6, a CCD camera 7 connected to the observation optical system 6, and a control unit. The control unit includes a monitor 9, a control computer 10, and an operation panel 8.

[0010] Inside the processing chamber, a Y-axis table 2 is installed on the bottom of the chamber. This Y-axis table 2 is movable in the Y direction. Specifically, a guide is installed on the underside of the Y-axis table 2. This guide is slidably connected to a guide rail installed on the bottom of the processing chamber. A ball screw is also connected to the underside of the Y-axis table 2. By operating this ball screw with a drive member such as a motor, the Y-axis table 2 can move along the guide rail (in the Y direction). The upper surface of the Y-axis table 2 is a mounting surface for the well plate 11, which will be described later. In other words, the Y-axis table 2 functions as a holder for the well plate 11, which is the object to which the liquid material (coating solution) is applied. The well plate 11 corresponds to the substrate 5 in Figure 3.

[0011] An X-axis table 1 is installed on a Y-axis table 2. The X-axis table 1 is positioned on a structure that straddles the Y-axis table 2 in the X direction. A movable body, to which the Z-axis table 3 is connected, is mounted on the X-axis table 1 so as to be movable in the X direction. The movable body is movable in the X direction, for example, using a ball screw. The X-axis table 1 is fixed to the bottom surface of the processing chamber via the above structure. Therefore, the Y-axis table 2 described above is movable in the Y direction relative to the X-axis table 1.

[0012] A Z-axis table 3 is installed on the mobile body connected to the X-axis table 1, as described above. An observation optical system 6 and a coating mechanism 4 are connected to the Z-axis table 3. The observation optical system 6 is used to observe the coating position on the well plate 11, which is the object to be coated. The CCD camera 7 converts the observed image into an electrical signal. The Z-axis table 3 holds these observation optical system 6 and coating mechanism 4 so that they can move in the Z direction.

[0013] The control computer 10 and operation panel 8 for controlling the Y-axis table 2, X-axis table 1, Z-axis table 3, observation optical system 6, and coating mechanism 4, as well as the monitor 9 attached to the control computer, are installed outside the processing room. The monitor 9 displays image data converted by the CCD camera 7 mentioned above, and output data from the control computer 10. The operation panel 8 is used to input commands to the control computer 10.

[0014] Figure 2 is a schematic diagram showing the coating mechanism of the coating apparatus shown in Figure 1. Referring to Figure 2, the coating mechanism 4 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 liquid container 21. The coating needle holder 20 is detachably attached to the movable base 35. In other words, the movable base 35, as a base body, detachably holds the coating needle holder 20.

[0015] In the coating mechanism 4, the servo motor 41 is installed so that its central axis extends in the direction along the Z direction shown in Figure 1. A cam 43 is connected to the rotation axis of the servo motor 41. The cam 43 is rotatable about the central axis of the servo motor 41. The cam 43 includes a central part connected to the rotation axis of the servo motor 41 and a flange part connected to one end of the central part. The upper surface of the flange part (the surface on the servo motor 41 side) is the cam surface. This cam surface is formed in an annular shape along the outer circumference of the central part and is also formed in a slope shape so that the distance from the bottom surface of the flange part varies. Specifically, the cam surface includes an upper flat region where the distance from the bottom surface is the greatest (thickest), a lower flat region located at a distance from this upper flat region, and a slope part that smoothly connects the upper flat region and the lower flat region. The lower flat region is the region where the distance from the bottom surface is the greatest (thinnest).

[0016] A bearing 44 is positioned so as to be in contact with the cam surface of the cam 43. A cam connecting plate 45 is connected to this bearing 44. In the cam connecting plate 45, one end connected to the bearing 44 and the other end opposite to it are fixed to a movable part 46. A movable base 35, which serves 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 liquid material to, for example, a well 12 of a well plate 11. The coating needle 24 is positioned to protrude from the coating needle holder 20 on the lower surface of the coating needle holder 20 (the lower side opposite to the side where the servo motor 41 is located). A coating liquid container 21 is positioned below the coating needle holder 20. The coating needle 24 is held in an inserted state within the coating liquid container 21.

[0017] A fixing pin is fixed to the movable part 46. The other fixing pin is fixed to the frame that holds the servo motor 41. A spring is installed to connect these fixing pins. Due to this spring, the movable part 46 is subjected to a force directed toward the coating liquid container 21. In addition, the force of this spring maintains the bearing 44 pressed against the cam surface of the cam 43.

[0018] Furthermore, the movable part 46 and the movable base 35 are connected to a linear guide installed on a frame that holds the servo motor 41, and are able to move along the Z direction.

[0019] In the coating mechanism 4 described above, the servo motor 41 is driven to rotate its rotation axis, thereby rotating the cam 43. As a result, the position of the bearing 44 in the Z direction, which is in contact with the cam surface of the cam 43, changes in accordance with the rotation of the servo motor 41's rotation axis. Then, in accordance with this change in the position of the bearing 44 in the Z direction, the movable part 46 and the movable base 35 move in the Z direction, thereby changing the position of the coating needle 24 in the Z direction. In other words, the coating needle 24 can be made to reciprocate in the Z direction. With this operation, when the coating needle 24 is in the upper Z direction, the tip of the coating needle 24 is immersed in the coating liquid container 21 containing the liquid material. In this state, the coating operation is performed by the coating needle 24 protruding downward from the tip hole at the bottom of the coating liquid container 21. With the liquid material attached to the tip of the coating needle 24, the tip of the coating needle 24 protrudes from the tip hole at the coating liquid container 21 and exits the coating liquid container 21. At this time, the liquid material is pulled upward by surface tension, and a nearly constant amount of liquid material adheres to the tip of the coating needle 24. This adhered liquid material is then transferred to the well plate 11, enabling a highly reproducible coating process.

[0020] For more details, refer to Figures 1 and 2. The coating speed command value output from the operation panel 8 is stored in the storage device of the control computer 10. During coating operation, the coating speed command value read from the storage device is transmitted to the control program of the coating mechanism 4. The control program of the coating mechanism 4 determines and rotates the servo motor 41 based on the coating speed command value. This causes the coating operation to be performed while the coating needle 24 reciprocates in the Z direction. If the control computer 10 is communicating with a higher-level control system (not shown), the coating speed command value may also be transmitted from the higher-level control system to the control program of the coating mechanism 4. In addition, parameters corresponding to the type of liquid material to be coated may be stored in the storage device of the control computer 10. The coating speed command value may be calculated according to the specified type of liquid material, coating amount, and coating dimensions.

[0021] Figure 3 is a schematic diagram showing the coating process using a coating needle. In Figure 3, the process proceeds in the order of (A), (B), and (C). In Figure 3, (A) shows the standby state before coating, (B) shows the coating state, and (C) shows the retracted state after coating. In the standby state (A), the tip 23 of the coating needle 24 is immersed in the coating liquid A (such as the mixed liquid A described later) in the coating liquid container 21. In the coating state (B), the coating needle 24 descends compared to the state in (A). In (B), the coating needle 24 penetrates the through hole 25 at the bottom of the coating liquid container 21, and the tip 23 comes into contact with the substrate 5 below it. The substrate 5 is an example of an object to be coated. The coating liquid A adhering to the tip 23 is transferred onto the substrate 5. As in (C), after the transfer is complete, the coating needle 24 rises, and the tip 23 is immersed in the coating liquid A again.

[0022] (Well plate) Figure 4 is a schematic perspective view of a well plate. Referring to Figure 4, in this embodiment, the coating liquid, as a liquid material, is supplied by coating into the interior of the multiple wells 12 formed on the well plate 11. However, the object to which the coating liquid is supplied is not limited to this. The well plate 11 has thickness in the Z direction, and multiple wells 12 are formed on its uppermost surface. The multiple wells 12 are recessed portions of the upper surface of the well plate 11. The multiple wells 12 may be spaced apart from each other, for example, in 8 rows in the Y direction and 12 rows in the X direction, for a total of 96 wells. The planar shape of the wells 12 can be arbitrary, such as a circle.

[0023] For example, the first coating solution containing cells, as described later, is supplied to multiple wells 12 of a well plate. Then, culture medium is supplied to the wells 12 to culture the cells. This allows for the creation of a cell tissue structure. The cell plate can be used for drug evaluation in drug efficacy and pharmacological evaluation and safety evaluation in the drug discovery process. The well plate 11 may be a 6-well plate with multiple wells 12, for example, 6 wells. However, the well plate 11 may also be a 12-well plate with 12 wells 12. Alternatively, the well plate 11 may be a 24-well plate, 48-well plate, 96-well plate, or 384-well plate with 24, 48, 96, or 384 wells 12. Alternatively, the cell tissue structure may be created by supplying the first coating solution to a dish plate or petri dish. Furthermore, a well plate with a single well 12 may also be used.

[0024] (Method for manufacturing cell tissue structures) Here, a method for producing a cell tissue structure according to this embodiment will be described. The cell tissue structure of this embodiment is obtained by coating a cell suspension containing the target cells with a culture vessel and culturing the target cells.

[0025] Figure 5 is a flowchart showing the method for producing a cell tissue structure according to this embodiment. As shown in Figure 5, the number of cells and the cell diameter are counted (S10). Here, the process of deriving the number of cells and the cell diameter is referred to as "counting". The number of cells is the number of cells contained in the cell suspension. "Cell suspension" means a liquid containing cells. A cell suspension is, for example, a liquid in which cultured cells or microorganisms are dispersed.

[0026] Next, the cell volume is calculated (S20). The cell volume is calculated from the count results of the cell number and cell diameter obtained in step (S10). The cell suspension containing the cells whose cell volume has been calculated is centrifuged. This yields a cell pellet (S30). The "cell pellet" is the precipitate obtained by centrifuging the cell suspension. Using this cell pellet, the cell pellet volume is calculated (S40). The "cell pellet volume" is the total volume of the cell pellet after the cell suspension has been centrifuged.

[0027] Subsequently, a solvent is added to the cell pellet, taking into account the cell volume fraction, to obtain a mixture (S50). In other words, the amount of solvent necessary for the cells in the mixture to have the desired cell volume fraction is added to the cell pellet prepared in step (S30). The amount of solvent required is calculated based on the volume of the cell pellet obtained in step (S40). The "solvent" is a biomaterial such as extracellular matrix. The "solvent" may also contain a thickening agent. The "cell volume fraction" will be explained later. The steps in Figure 5 will be explained in detail below.

[0028] First, let's explain step (S10). Figure 6 is a chart listing the processes performed in step (S10) of Figure 5. As shown in Figure 6, in step (S10) for counting cell diameters, the average value of the diameters of the cells contained in the cell suspension is calculated as the cell diameter (S11). The cell diameter is, for example, the average value (average cell diameter) of the diameters of the cells contained in the cell suspension. Here, the cell diameter is defined as the average cell diameter.

[0029] In step (S10), the cell count is measured using an automated cell counter. The automated cell counter is a device that measures cell count, cell diameter, cell viability, etc. A hemocytometer may be used instead of the automated cell counter. A hemocytometer is a device that calculates cell count and cell viability. Both the automated cell counter and the hemocytometer calculate cell count and cell viability using a microscope and visual inspection.

[0030] The average cell diameter is calculated from values ​​measured by an automated cell counter. Alternatively, the average cell diameter may be calculated by image analysis of phase-contrast microscope images. This allows for an accurate determination of the average cell diameter.

[0031] Depending on the model, an automated cell counter may measure the average cell diameter of both living cells and dead cells in a cell suspension. In this case, the overall average cell diameter may not be measured depending on the model. When counting the average cell diameter of living cells and dead cells in this way, the process shown in Figure 6 (S12) may be performed. That is, the average cell diameter is calculated as the average of the diameters of all cells, which are calculated by weighted averaging from the number of living cells and dead cells contained in the cell suspension. The number of living cells is the number of living cells. The number of dead cells is the number of dead cells. In this case, the average cell diameter of all cells (the average value of the diameters of all cells) can be obtained using the following formula (1).

[0032]

number

[0033] The meaning of the symbols in each term of the above formula is as follows: D represents the average cell diameter (unit: μm). l indicates the average cell diameter of living cells (in μm). r indicates the cell viability (in %). d This represents the average cell diameter (in μm) of dead cells. For example, if the average cell diameter of living cells is 15 μm, the average cell diameter of dead cells is 10 μm, and the cell viability is 80%, then by applying this to equation (1) above, the average value D of the diameter of all cells is 14 μm. In this way, even when using a machine that calculates the average cell diameter of living and dead cells separately, the average value of the diameter of all cells can be obtained by weighted averaging.

[0034] In addition, the average value of the diameters of all cells in a cell suspension in which multiple types of cells are mixed can be calculated by weighted average in the same manner as described above. Here, the average value of the diameters of all cells also means the average cell diameter of all cells. That is, when there are two types of cells, A cells and B cells, the average cell diameter of all cells including them is obtained by the following formula (2).

[0035] [Number]

[0036] The meanings of the symbols of each term in the above mathematical formula are as follows. D represents the average cell diameter (unit: μm). D a represents the average cell diameter of A cells (unit: μm). r a represents the mixing ratio of A cells in the liquid in which multiple cells are mixed (unit: %). D b represents the average cell diameter of B cells (unit: μm). r b represents the mixing ratio of B cells in the liquid in which multiple cells are mixed (unit: %). For the overall average cell diameter D in a liquid in which three or more types of cells are mixed, it is also obtained by the above formula (2). That is, the product of the average cell diameter and the mixing ratio of each cell after the third type is added to the numerator of the above formula (2). Thereby, the average cell diameter D is obtained.

[0037] The mixing ratio of each of the above cells is the ratio of the number of cells of each cell to the total number of cells after mixing. That is, for example, the mixing ratio r a (unit: %) of A cells in a cell suspension in which two types, A cells and B cells, are mixed is obtained by the following formula (3).

[0038] [Number]

[0039] The meanings of the symbols of each term in the above mathematical formula are as follows. A n is the number of A cells in the liquid (unit: cells). B nThis represents the number of B cells in the solution (unit: cells).

[0040] The above describes the calculation of the average diameter of cells in a cell suspension in step (S10), as in step (S11). However, as shown in Figure 6, as another example, in step (S10) for counting cell diameters, the median diameter of the cells contained in the cell suspension may be used as the cell diameter (S13). If the distribution of cell diameters deviates from a normal distribution, the median or mode of cell diameters may be used instead of the average cell diameter. If the distribution of cell diameters deviates from a normal distribution, this method yields a cell tissue structure with higher stability and reproducibility compared to using the average cell diameter.

[0041] As shown in Figure 6, in step (S10), cells to be excluded from the counting of cell diameters may be selected (S14). In other words, among the cells in the cell suspension, those with a diameter exceeding the maximum diameter and those with a diameter less than the minimum diameter are excluded from the counting of cell diameters in step (S10). The maximum and minimum diameters mentioned above may be predetermined values. Alternatively, the measurement limits of the measuring device may be set as the maximum and minimum diameters, and cells with diameters outside the range of diameters that the device can measure may be excluded from the counting. This allows the average cell diameter, median cell diameter, etc., to be calculated without including diameters as outliers. As a result, the reliability of calculated values ​​such as the average cell diameter and median cell diameter is improved. This results in a cell tissue structure with high stability and reproducibility. For example, 20 μm, 25 μm, or 30 μm may be used as the maximum value. For example, 3 μm, 5 μm, or 8 μm may be used as the minimum value.

[0042] For example, when calculating the average cell diameter using an automated cell counter, the automated cell counter may have a function to set an outlier threshold. This function can be used to set a threshold, and values ​​exceeding the maximum value and values ​​below the minimum value based on that threshold may be excluded. When using phase-contrast microscopy images and image analysis, the average cell diameter may be calculated after excluding cells with values ​​below and above the threshold from the cell diameter distribution obtained from the image analysis.

[0043] Next, we will explain step (S20). The cell volume can be calculated from the number of cells and the cell diameter (e.g., average cell diameter) using the following formula (4). This calculation can be done manually or by computer.

[0044]

number

[0045] The symbols in the above formula have the following meanings: V represents cell volume (unit: μL). D represents average cell diameter (unit: μm). n represents the number of cells (unit: cells). In other words, cell volume can be roughly calculated by multiplying the average volume of a single cell by the number of cells.

[0046] Next, steps (S30) and (S40) will be described. When preparing a cell pellet from a cell suspension, for example, the cell suspension is placed in a microtube with a volume of 1.5 mL and centrifuged. Centrifugation yields a cell pellet in the microtube. The microtube is marked with a marker pen at the height of the cell pellet. The height of the cell pellet refers to the uppermost part of the substance in the microtube after centrifugation, excluding the supernatant. Next, the cell suspension in the microtube is mixed again to return it to a state where the supernatant and cell pellet are not separated. Then, in order to empty the microtube, all of the cell suspension in the microtube is transferred to another container. After the microtube is empty, water is added to the microtube up to the position of the previously made mark. The volume of water added at this time is measured. The measured volume of water is determined as the volume of the cell pellet.

[0047] If the cell pellet volume is determined, the cell volume ratio α can be calculated using the following equation (5).

[0048]

number

[0049] In equation (5) above, V p V represents the cell pellet volume (μL). V represents the cell volume (unit: μL) calculated earlier.

[0050] When conducting experiments under identical conditions, the cell volume ratio α can be considered constant. Here, identical conditions mean suspending the same type of cells in the same liquid and performing centrifugation under identical conditions. In this case, the cell pellet volume V p It is produced as the product of the cell volume ratio α and the cell volume.

[0051] Next, we will explain step (S50). The cell pellet volume obtained in step (S40) is the sum of the volume of cells contained in the cell suspension after centrifugation and the volume of liquid contained in the substance after centrifugation. This liquid volume is the volume of the liquid that makes up the cell suspension, excluding the portion that was separated into supernatant by centrifugation.

[0052] The cell volume fraction is expressed by the following formula (6). This calculation can be done manually or by computer.

[0053]

number

[0054] The meanings of the symbols in the above formula are as follows: φ represents the cell volume fraction (unit: %). V represents the volume of cells contained in the cell pellet after centrifugation (unit: mL). l V represents the volume (in mL) of the liquid (excluding the supernatant) contained in the cell pellet after centrifugation. L represents the volume (in mL) of the solvent mixed with the cell pellet in step (S50) to obtain the mixture. Therefore, V l +V corresponds to the volume of the cell pellet after centrifugation.

[0055] The mixture is the final substance obtained from the cell pellet. The mixture is supplied by application or other means for the preparation of cell tissue structures. To achieve the desired cell volume fraction in the mixture, a solvent containing biomaterials is added to the cell pellet. Here, the cell volume fraction α is a value that can be arbitrarily set by the operator. (V = cell volume in cell pellet, V = liquid volume in cell pellet) l This has already been determined by process (S40). Therefore, the amount of solvent L to be added can be uniquely determined from equation (6) above. The amount of solvent L is calculated by hand or by computer. By controlling the amount of solvent L, the cell volume fraction α is controlled to the desired value. The mixture obtained above is used as the first coating solution described later, especially when supplied by a coating device having a coating needle.

[0056] Based on the cell volume fraction, cell volume, and cell pellet volume described above, the total volume after bioink preparation can be calculated using the following formula (7). In this embodiment, the bioink corresponds to the first coating solution described later, especially when a coating device is used.

[0057]

number

[0058] The meaning of each symbol in the above formula is as follows: BI V This is the total volume (in μL) of the bioink after preparation. The cell volume V and cell volume fraction α in the cell pellet are the same as above. In other words, the total volume of the bioink after preparation allows us to determine the total volume of the mixture after adding the solvent to the cell pellet.

[0059] Furthermore, the volume of solvent (in μL) used to obtain the mixture for preparing the bio-ink can be calculated using the following formula (8). The symbols in each term have the same meaning as above.

[0060]

number

[0061] (Effects and Benefits) In the method for producing a cell tissue structure according to this disclosure, the number of cells in the cell suspension and the cell diameter are counted (S10). The cell volume is calculated from the number of cells and cell diameter obtained in the above counting step (S20). A cell pellet is obtained by centrifugation of the cell suspension containing the cells whose cell volume has been calculated (S30). The cell pellet volume, which is the total volume of the cell pellet, is calculated (S40). Based on the cell pellet volume, a solvent containing biomaterials is added to the cell pellet so that the cells are in a desired cell volume fraction, and a mixture is obtained (S50).

[0062] In this way, the cell volume fraction, which is the volume percentage of cells themselves in the mixture supplied by coating or other means for the creation of cell tissue structures, is controlled. The density of cells in the cell tissue structure formed using that mixture is determined by the level of the cell volume fraction.

[0063] Conventionally, only the number of cells in a cell tissue structure was considered, and cell volume, cell concentration (cell number concentration), and cell volume fraction were not taken into account. In this case, variations occur in the volume fraction of cells in the mixed solution supplied by coating or other means. However, according to this disclosure, by controlling the cell volume fraction to a desired value, the cell volume fraction becomes constant. Therefore, variations in the volume fraction of cells in all cell tissue structures produced are eliminated. Consequently, cell tissue structures with high stability and reproducibility can be provided. [Examples]

[0064] Example 1 was conducted as a first preliminary experiment. In the first preliminary experiment, the effects of cell volume and cell volume fraction on the density of the cell tissue structure were investigated. First, the method for manufacturing the cell tissue structure, which is common to all subsequent examples, will be explained using Figures 7 to 15. The manufacturing method shown here is a general method for supplying the first coating solution using a coating device having a coating needle.

[0065] Figure 7 is a schematic diagram showing the state before the first coating solution is applied in the embodiment. Referring to Figure 7, first the tip of the coating needle 24, which constitutes the coating device 100 (see Figure 1), is immersed in the first coating solution A, and the first coating solution A adheres to the tip of the coating needle 24. Figure 8 is a schematic diagram showing the composition of the first coating solution in the embodiment. Referring to Figure 8, the first coating solution A is obtained by mixing cells C to be cultured with a first solvent m. The first solvent m corresponds to the "solvent" mentioned above and includes, for example, collagen. The cells C in Figure 8 include C1 and C2, which will be described later. Figure 9 is a schematic diagram showing the process of applying the first coating solution in the embodiment. Referring to Figure 9, the tip of the coating needle 24 to which the first coating solution A is attached comes into contact with, for example, the bottom (well bottom 12a) of the well 12 (see Figure 3). This is done by the coating needle 24 to which the first coating solution A is attached moves downward as shown by arrow M1 in Figure 7. This applies the first coating liquid A to the bottom 12a of the well. Figure 10 is a schematic diagram showing the state after the first coating liquid has been applied in this embodiment. Referring to Figure 10, the coating needle 24 then moves upward as indicated by arrow M2. In this way, the first coating liquid A is applied to a container such as the well 12 using a so-called pin-type coating device 100.

[0066] Figure 11 is a schematic diagram showing the process of supplying the second coating solution in the embodiment. Figure 12 is a schematic diagram showing the state of the well after the process in Figure 11 has been performed. Referring to Figures 11 and 12, the second coating solution B is supplied into the well 12 so as to cover the first coating solution A that has been applied into the well 12. The second coating solution B may be dispensed by drop, for example, a dispenser, but the method of supplying the second coating solution B is not limited to this. The second coating solution B may be supplied by any of the following methods selected from the group consisting of a pin method, an inkjet method, a dispenser method, and manual supply using a pipette.

[0067] Figure 13 is a schematic diagram showing the process of supplying the culture medium in the embodiment. Referring to Figure 13, after the step of supplying the second coating solution B, the culture medium M is supplied into the well 12. The culture medium M is supplied so as to immerse and cover the first coating solution A and the second coating solution B. The method of dropping the culture medium M is not particularly limited. The culture medium M may be supplied by any of the group consisting of a pin method, an inkjet method, a dispenser method, or manual dispensing using a pipette. Alternatively, the culture medium M may be supplied by a dispenser or a micropump.

[0068] Figure 14 is a schematic diagram showing the state of cells in the first coating solution before culture. Figure 15 is a schematic diagram showing the state of cells in the first coating solution after culture. Referring to Figures 14 and 15 (and Figure 8), cell C in the first coating solution A before culture contains cell C1 and cell C2. Through culture, both cell C1 and cell C2 grow from the state in Figure 14 to the state in Figure 15, and a cell tissue structure is formed.

[0069] In the manufacturing method shown in Figures 7 to 15, for example, in Figures 7 to 10, the mixed solution A is stored in a coating solution container 21 having a through hole at the bottom. The coating needle 24 penetrates the coating solution container 21 containing the mixed solution A. This transfers the mixed solution A to the bottom of the well 12a, which is the object to be coated. This allows for a stable supply of the first coating solution. Therefore, by subsequently adding the second coating solution and culture medium as shown in Figures 11 to 15, a cell tissue structure with high stability and reproducibility can be produced.

[0070] In this example, the first coating solution was a mixture of cells constituting the cell tissue structure to be formed, thrombin, and sodium hyaluronate. Thrombin is a gelling initiator for the gel used to create the cell tissue structure. Sodium hyaluronate is a thickening agent for the mixture. The second coating solution was a mixture containing fibrinogen and sodium hyaluronate. Fibrinogen is a gelling agent. Sodium hyaluronate is a thickening agent for the mixture. The cell tissue structure was formed by the manufacturing method shown in Figures 7 to 15.

[0071] In this example, two types of first coating solutions were prepared. One contained iPS cardiomyocytes as cells. The other contained HepG2 cells, which are cancer cells. The number of iPS cardiomyocytes and HepG2 cells contained in the coating solution was 1 × 10⁶ in both cases. 7 They were cells.

[0072] Two types of first coating solutions were centrifuged. The average cell diameter of the cells contained in each first coating solution and the cell pellet volume immediately after centrifugation (cell count 1 × 10⁻¹⁶) were determined. 7 The values ​​per cell were as shown in Table 1.

[0073] [Table 1]

[0074] Each of the first coating solutions was mixed with the same amount and concentration of sodium hyaluronate (0.55 mg / mL). The two prepared first coating solutions were applied, and then the second coating solution and culture medium were added. This created cell tissue structures.

[0075] Figure 16 shows photographs of the cell tissue structures prepared in Example 1. The photograph on the left of Figure 16 is a cell tissue structure obtained from the first coating solution of iPS cardiomyocytes. The photograph on the right of Figure 16 is a cell tissue structure obtained from the first coating solution of HepG2 cells. As shown in Figure 16, the cell tissue structure made from iPS cardiomyocytes had more gaps and lower density compared to the cell tissue structure made from HepG2 cells. This is because iPS cells have a smaller average cell diameter than HepG2 cells, so even with the same number of cells, the cell volume fraction in the mixture decreased due to the mixing of sodium hyaluronate. From Table 1, the cell pellet volume of iPS cardiomyocytes was approximately 1 / 4 to 1 / 3 of the cell pellet volume of HepG2 cells.

[0076] From the above, it was found that even with the same number of cells, mixing the same amount and concentration of biomaterial and solvents such as thickeners results in different cell densities in the resulting cell tissue structures depending on the cell type. It was also found that for cell types, smaller cell diameters result in smaller cell volumes, leading to a decrease in the cell volume fraction in the mixture and a structure with many gaps, as shown in the left photograph of Figure 16. In other words, it was found that the cell volume fraction of the first coating solution needs to be individually set based on the cell volume calculated from the number of cells and cell diameter (average cell diameter, median cell diameter, etc.). [Examples]

[0077] Example 2 was conducted as a second preliminary experiment. In the second preliminary experiment, an investigation was conducted to compare the cell volume ratio α between different cell types. For three types of cells, the relationship between cell volume and cell pellet volume was compared. Cell volume was calculated from the number of cells and the average cell diameter in the mixture containing each cell type. The number of cells was counted using a cell counter. Cell pellet volume was measured after centrifuging the mixture in which the cell volume was measured and removing the supernatant. The cell volume ratio α was calculated from the cell volume and cell pellet volume. The three types of cells were human dermal fibroblasts A (NHDF-A), human dermal fibroblasts B (NHDF-B), and cancer cells (HepG2). The results are shown in Table 2.

[0078] [Table 2]

[0079] Table 2 shows that the cell volume ratio α, which is the ratio of cell pellet volume to cell volume, is between 2.7 and 2.9 for all cell types. When calculating the cell pellet volume and cell volume fraction from the cell volume, the cell volume ratio α shown in Table 2 may be used. Alternatively, the cell pellet volume may be measured using the method described above, such as using a microcentrifuge tube.

[0080] The values ​​shown in Table 2 serve as a guideline for the cell volume ratio α. As mentioned earlier, when experiments are conducted under identical conditions, i.e., identical centrifugation conditions, the cell volume ratio α can be considered to be a constant value. Therefore, under identical conditions, the known value of the cell volume ratio α can be used directly. However, the cell volume ratio α is a value that changes depending on conditions such as gravity during centrifugation. For this reason, if it is necessary to consider conditions such as gravity, it is preferable to measure the cell volume ratio α each time. [Examples]

[0081] Example 3 was conducted as the first main experiment. In the first main experiment, an example of preparing a cell tissue structure in which the cell volume fraction of the first coating solution was made much higher than usual is shown.

[0082] As the first coating solution, a liquid in which cardiomyocytes were dispersed in collagen, which was used as the solvent, was applied. Collagen was added as an extracellular matrix, which is a biomaterial. Collagen was also added as a gelling agent. iPS cell-derived cardiomyocytes and human cardiac fibroblasts were used as cardiomyocytes. The ratio of viable cells between iPS cell-derived cardiomyocytes and human cardiac fibroblasts was 4:1.

[0083] iPS cell-derived cardiomyocytes have a combined cell count of 5.04 × 10⁶ (including both living and dead cells). 6 The average cell diameter of the cells was 13.9 μm. Of these, the number of viable cells was 4.29 × 10⁶. 6 The average cell diameter was 14.7 μm. The number of dead cells was 7.53 × 10⁶. 5 The average cell diameter of the cells was 9.18 μm. The overall cell viability of iPS cell-derived cardiomyocytes was 85%. The human cardiac fibroblasts had a total cell count of 5.94 × 10⁶ cells, including both living and dead cells. 6 The average cell diameter of the cells was 18.0 μm. Of these, the number of viable cells was 5.73 × 10⁶. 6 The average cell diameter was 18.18 μm. The number of dead cells was 2.04 × 10⁶. 5The average cell diameter of the cells was 12.84 μm. The overall cell viability of human cardiac fibroblasts was 96%. The total amount of iPS cell-derived cardiomyocytes was 18.7% (1.07 × 10⁻¹⁰) of the total amount of human cardiac fibroblasts. 6 Cells were added. This adjusted the ratio of living cells of the two types to approximately 4:1. The number of cells after mixing the two types was 6.15 × 10⁶. 6 These were cells, and the average cell diameter was 14.6 μm.

[0084] The cell volume of the first coating solution was 10.1 μL, and the cell pellet volume was 28.2 μL. The cell volume ratio α was 2.8. The first coating solution was centrifuged under conditions of 300 G gravity for 5 minutes. The total volume of the first coating solution after bioink preparation was 40.3 μL, and the volume of the biomaterial "solvent" was 12.1 μL.

[0085] The collagen concentration before the addition of cardiomyocytes was 2.1 mg / mL, and the final concentration after the addition of cardiomyocytes was 0.63 mg / mL. Therefore, the final cell volume fraction of the first application solution was determined to be 25%. The cell number concentration of the first application solution was 1.53 × 10⁻⁶. 8 The concentration was set to cells / mL. In addition to collagen, phosphate-buffered saline (+) (PBS (+)) was added as a solvent to the first coating solution. This first coating solution was automatically applied to the object to be coated using a coating needle with a circular cross-section of 1 mm in diameter.

[0086] A second coating solution containing 0.75% methylcellulose was added to cover the first coating solution. Approximately 15 μL of methylcellulose was added as a thickening agent. Phosphate-buffered saline (+) (PBS (+)) was added to the second coating solution as a solvent. The second coating solution was automatically added by a dispenser. The second coating solution was added within 5 seconds after the first coating solution was applied. After the addition of the second coating solution, the culture medium was manually added using a micropipette. As a result, a cell tissue structure was formed.

[0087] Figure 17 is a photograph showing the phase-contrast observation results of the cell tissue structure prepared in Example 3. As shown in Figure 17, by using the first coating solution with a high cell volume fraction, a cardiomyocyte tissue with high cell density was produced. The cell tissue structure shown in Figure 17 has a much higher cell density than a typical cell tissue structure. In this example, it was verified that a structure with high cell density can actually be produced by controlling the cell volume ratio to its upper limit.

[0088] Figure 18 shows the phase-contrast observation results of 50 cardiomyocyte tissue structures fabricated in a single 96-well plate. As shown in Figure 18, numerous cardiomyocyte tissue structures were fabricated stably and with high reproducibility. In other words, all 50 tissue structures were fabricated with similarly high cell density and without significant variation.

[0089] Furthermore, although not shown in the figures, the following results were obtained in this embodiment. In this embodiment, when the diameter of the first coating solution was 1000 μm, it was confirmed that the cells inside the coating solution had a thickness of approximately 50 μm to 150 μm and were densely stacked. Thus, a high-density three-dimensional cell tissue was created in this embodiment. Furthermore, cell viability was confirmed by staining of the cell nucleus, actin, and troponin. This confirmed the expression of actin and troponin, which are characteristic of cardiomyocytes. [Examples]

[0090] Example 4 was conducted as the second main experiment. In the second main experiment, the effect of using a first coating solution with a lower cell volume fraction and relatively smaller cell diameter than in the first main experiment was evaluated on the preparation of cell tissue structures.

[0091] As the first coating solution, a liquid in which PC-12 cells were dispersed in collagen, which was the solvent, was applied. Collagen was added as an extracellular matrix, which is a biomaterial.

[0092] PC-12 has a total of 2.88 × 10⁶ cells, including both living and dead cells. 6The average cell diameter of the cells was 8.55 μm. Of these, the number of viable cells was 2.2 × 10⁶. 6 The average cell diameter was 8.54 μm. The number of dead cells was 6.8 × 10⁶. 5 The average cell diameter was 8.58 μm. The overall cell viability of PC-12 was 76%.

[0093] The cell volume of the first coating solution was 0.94 μL, and the cell pellet volume was 2.64 μL. The cell volume ratio α was 2.8. The first coating solution was centrifuged under conditions of 300 G gravity for 5 minutes. The total volume of the first coating solution after bioink preparation was 18.8 μL, and the volume of the biomaterial "solvent" was 16.2 μL.

[0094] The collagen concentration before the addition of PC-12 was 0.73 mg / mL, and the final concentration after the addition of PC-12 was 0.63 mg / mL. Therefore, the cell volume fraction of the first application solution was ultimately determined to be 5%. The cell number concentration of the first application solution was 1.52 × 10⁻⁶. 8 The concentration was set to cells / mL. In addition to collagen, phosphate-buffered saline (+) (PBS (+)) was added as a solvent to the first coating solution. This first coating solution was automatically applied to the object to be coated using a coating needle with a circular cross-section diameter of 0.5 mm.

[0095] A second coating solution containing 0.75% methylcellulose was added to cover the first coating solution. Approximately 15 μL of methylcellulose was added as a thickening agent. Phosphate-buffered saline (+) (PBS (+)) was added to the second coating solution as a solvent. The second coating solution was automatically added by a dispenser. The second coating solution was added within 5 seconds after the first coating solution was applied. After the addition of the second coating solution, the culture medium was manually added using a micropipette. As a result, a cell tissue structure was formed.

[0096] Figure 19 is a photograph showing the phase-contrast observation results of the cell tissue structure prepared in Example 4. As shown in Figure 19, we were able to prepare PC-12 cell tissue with an appearance similar to that of a planar cultured structure.

[0097] Although the cell number concentration is almost the same in Example 3 and Example 4, the cell diameter is larger in Example 3 than in Example 4. Therefore, the cell volume fractions of Example 3 and Example 4 are significantly different. The cell volume fraction of Example 3 is five times that of Example 4. As a result, the density of cell tissue structures in Example 3 is higher than in Example 4 in the phase-contrast microscope images. [Examples]

[0098] Example 5 was conducted as the third main experiment. In the third main experiment, the effect of using a first coating solution with a lower cell volume fraction than the first main experiment and a larger cell diameter than the second main experiment on the preparation of cell tissue structures was evaluated.

[0099] As the first coating solution, a liquid in which human cardiac fibroblast cells were dispersed in methylcellulose, which was the solvent, was applied. Methylcellulose was added as both a biomaterial and a thickening agent.

[0100] Human cardiac fibroblasts have a combined total of 0.97 × 10⁻¹⁴ cells (living and dead cells). 6 The average cell diameter of the cells was 18.80 μm. Of these, the number of viable cells was 0.94 × 10⁶. 6 The average cell diameter was 18.91 μm. The number of dead cells was 0.97 × 10⁶. 6 The average cell diameter was 15.43 μm. The overall cell viability of PC-12 was 97%.

[0101] The cell volume of the first coating solution was 3.37 μL, and the cell pellet volume was 9.45 μL. The cell volume ratio α was 2.8. The first coating solution was centrifuged under conditions of 200 G gravity for 5 minutes. The total volume of the first coating solution after bioink preparation was 13.50 μL, and the volume of the biomaterial "solvent" was 4.05 μL.

[0102] The concentration of methylcellulose before the addition of human cardiac fibroblasts was 0.76 mg / mL, and the final concentration after the addition of human cardiac fibroblasts was 0.65 mg / mL. Therefore, the final cell volume fraction of the first application solution was determined to be 5%. The cell number concentration of the first application solution was 1.44 × 10⁻⁶. 7 The concentration was set to cells / mL. In addition to methylcellulose as a solvent, phosphate-buffered saline (+) (PBS (+)) was added to the first coating solution. This first coating solution was automatically applied to the object to be coated using a coating needle with a circular cross-section of 0.5 mm in diameter.

[0103] A second coating solution containing 0.75% methylcellulose was added to cover the first coating solution. Approximately 15 μL of methylcellulose was added as a thickening agent. Phosphate-buffered saline (+) (PBS (+)) was added to the second coating solution as a solvent. The second coating solution was automatically added by a dispenser. The second coating solution was added within 5 seconds after the first coating solution was applied. After the addition of the second coating solution, the culture medium was manually added using a micropipette. As a result, a cell tissue structure was formed.

[0104] Figure 20 is a photograph showing the phase-contrast observation results of the cell tissue structure prepared in Example 5. As shown in Figure 20, we were able to prepare a cell tissue of human cardiac fibroblasts that had an appearance similar to that of a planar cultured structure.

[0105] The cell number concentration differs significantly between Example 4 and Example 5. The cell number concentration in Example 4 is approximately 10 times that of Example 5. However, the cell volume fraction is the same in Example 4 and Example 5. Therefore, the external appearance, i.e., density, of the cell tissue structure is almost the same between Example 4 and Example 5.

[0106] (Effects of the Examples) Based on the above examples, in the method for producing cell tissue structures according to this disclosure, it is preferable that the cell volume fraction of cells contained in the cell suspension is 5% or more and 35.7% or less. This makes it possible to produce cell tissue structures with high stability and reproducibility. The cell volume fraction may be 25% or more.

[0107] From the above examples, the method for producing cell tissue structures according to this disclosure results in a cell number (cell number concentration) in the cell suspension of 1.44 × 10⁻⁶. 7 It is preferable that the cell count is 1.5 × 10¹⁶ cells / mL or higher. This allows for the production of cell tissue structures with high stability and reproducibility. The cell count (cell concentration) is 1.5 × 10¹⁶ cells / mL or higher. 7 cells / mL or more 1×10 10 The cell count may be less than or equal to 1.0 × 10⁻⁶ cells / mL. Alternatively, the cell count (cell concentration) may be 1.0 × 10⁻⁶. 7 The cell count (cell concentration) may be 1.0 × 10⁻⁶ or higher. 7 When cells / mL is greater than 1 × 10 10 It may be less than cells / mL.

[0108] (Possible modifications in each embodiment) In Example 1, the gelling initiator in the first coating solution may be thrombin, and the gelling agent in the second coating solution may be fibrinogen. However, in each example, the gelling initiator in the first coating solution may be calcium chloride, and the gelling agent in the second coating solution may be sodium alginate. The gelling initiator in the first coating solution may be calcium chloride, and the gelling agent in the second coating solution may be carrageenan. The gelling initiator in the first coating solution may be an alcohol, and the gelling agent in the second coating solution may be tamarind seed gum.

[0109] In Example 1, the first coating solution contains cells and a gelling initiator, and the second coating solution contains a gelling agent. However, it is not limited to this. Except for the example in which cardiomyocytes are used as the cells, the first coating solution may contain cells and a gelling agent, and the second coating solution may contain a gelling initiator. When the first coating solution contains a gelling agent and the second coating solution contains a gelling initiator, a cell-embedded gel is formed in a short time. As a result, it is possible to produce cell tissue that conforms more closely to the desired shape, especially the desired planar shape, due to its high shape stability.

[0110] In Example 1, sodium hyaluronate is mixed into the first coating solution as a thickening agent. However, it is not limited to this. Other thickening polysaccharides such as sodium alginate may be used as the thickening agent for the first coating solution. The thickening agent is effective in improving the shape retention of the coating solution after application. In other words, the thickening agent suppresses the coating solution from flowing out and losing its shape after application, causing it to combine with other adjacent coating solutions, and thus maintains the independence of the cell tissue. Sodium hyaluronate, in particular, has high adhesive properties. Sodium hyaluronate adheres well to the coating solution container 21 and the cells, making it optimal for improving the shape retention of the coating solution. Furthermore, sodium hyaluronate is of biological origin and has the advantage of being highly compatible with cell proliferation. The thickening agent may also be mixed into the second coating solution.

[0111] In Example 3, collagen, a temperature-responsive gelling agent, was used as the first coating solution. Methylcellulose was used as the second coating solution. However, the examples are not limited to these. In each example, Matrigel, gelatin, or GelMA (gelatin methacryloyl) may be used as the first coating solution. Alternatively, a mixture containing collagen and at least one of Matrigel, gelatin, or GelMA (gelatin methacryloyl) may be used as the first coating solution. At least one selected from the group consisting of cellulose, cellulose nanofibers, chitin, chitosan, chitin nanofibers, chitosan nanofibers, carboxymethylcellulose, hydroxybutylcellulose, sodium alginate, sodium hyaluronate, polyethylene glycol, gellan gum, carrageenan, pectin, xanthan gum, gelatin, agarose, and polyvinyl alcohol may be used as the second coating solution.

[0112] Examples 1 to 5 describe a method for producing a cell tissue structure by applying a first coating solution using a coating device 100 and a coating needle 24. However, the method used to produce the cell tissue structure in these embodiments and examples is not limited to these. In these embodiments and examples, instead of a coating device with a coating needle, a medium equivalent to the first coating solution used when the coating needle 24 is applied may be supplied by an inkjet device or a dispenser to produce the cell tissue structure. When using these, there is no distinction between the first and second coating solutions. The supplied mixture contains the desired cells and a gelling agent, etc. Inkjet devices are capable of fine coating and offer excellent high speed. Dispensers can be used for a wider range of liquid materials than inkjet devices. Dispensers are suitable for coating relatively large quantities. Coating using a coating device with a coating needle eliminates the possibility of nozzle clogging. Coating with a coating needle can be used for a wide range of liquid materials. Furthermore, a coating needle allows for fine coating. As described above, various devices may be used or combined, taking advantage of the characteristics of each type of coating device. This makes it possible to efficiently produce high-quality cell tissue.

[0113] The features described in the embodiments above may be applied in appropriate combinations to the extent that they do not contradict the technical standards.

[0114] The embodiments disclosed herein should be considered in all respects to be illustrative and not restrictive. The scope of the present invention is indicated by the claims rather than by the foregoing description, and all modifications within the meaning and scope equivalent to the claims are intended to be included.

[0115] (Note) The various aspects of this disclosure are summarized below as an appendix.

[0116] (Note 1) A process of counting the number of cells contained in a cell suspension and the cell diameter, A step of calculating the cell volume from the number of cells and the cell diameter obtained in the counting step, A step of obtaining a cell pellet by centrifuging the cell suspension containing the cells whose cell volume has been calculated, A step of calculating the cell pellet volume, which is the total volume of the cell pellet, A method for producing a cell tissue structure, comprising the steps of: adding a solvent containing a biomaterial (for example, a biomaterial and a thickener) to the cell pellet based on the volume of the cell pellet, so that the cells are in a desired cell volume fraction, in order to obtain a mixed solution.

[0117] (Note 2) A method for producing a cell tissue structure according to Appendix 1, wherein the step of counting the cell diameters is to determine the average value of the diameters of the cells contained in the cell suspension.

[0118] (Note 3) A method for producing a cell tissue structure as described in Appendix 2, wherein the average value of the diameter of all cells, calculated by weighted averaging from the number of living cells and the number of dead cells contained in the aforementioned cells, is used.

[0119] (Note 4) A method for producing a cell tissue structure according to Appendix 1, wherein the step of counting the cell diameters is to determine the median diameter of the cells contained in the cell suspension.

[0120] (Note 5) A method for producing a cell tissue structure according to any one of the appendices 1 to 4, wherein, in the step of counting the cell diameter, cells having a diameter exceeding the maximum diameter and cells having a diameter less than the minimum diameter are excluded from the counting of the cell diameter in the counting step.

[0121] (Note 6) A method for producing a cell tissue structure according to any one of the appendices 1 to 5, wherein the cell volume fraction of the cells contained in the cell suspension is 5% or more and 35.7% or less.

[0122] (Note 7) The cell number concentration in the aforementioned cell suspension is 1.5 × 10⁻⁶. 7 cells / mL or more 1×10 10 A method for producing a cell tissue structure as described in any one of the appendices 1 to 6, wherein the cell density is less than or equal to cells / mL.

[0123] (Note 8) The steps include storing the aforementioned mixture in a coating liquid container having a through hole at the bottom, A method for producing a cell tissue structure according to any one of the appendices 1 to 7, further comprising the step of transferring the mixture to an object to be coated by having a coating needle penetrate the coating container which contains the mixture. [Explanation of Symbols]

[0124] 1 X-axis table, 2 Y-axis table, 3 Z-axis table, 4 coating mechanism, 5 substrate, 6 observation optics, 7 CCD camera, 8 operation panel, 9 monitor, 10 control computer, 11 well plate, 12 wells, 12a well bottom, 20 coating needle holder, 21 coating solution container, 23 tip, 24 coating needle, 25 through hole, 35 movable base, 41 servo motor, 43 cam, 44 bearing, 45 cam connecting plate, 46 movable part, A coating solution (first coating solution: mixed solution), B second coating solution, C cells, C1 first cells, C2 second cells, M culture medium, m first solvent.

Claims

1. A process of counting the number of cells contained in a cell suspension and the cell diameter, A step of calculating the cell volume from the number of cells and the cell diameter obtained in the counting step, A step of obtaining a cell pellet by centrifuging the cell suspension containing the cells whose cell volume has been calculated, A step of calculating the cell pellet volume, which is the total volume of the cell pellet, A method for producing a cell tissue structure, comprising the steps of: adding a solvent containing a biomaterial to the cell pellet based on the volume of the cell pellet, so that the cells are in a desired cell volume fraction, in order to obtain a mixed solution.

2. The method for producing a cell tissue structure according to claim 1, wherein the step of counting the cell diameters is to determine the average value of the diameters of the cells contained in the cell suspension.

3. A method for producing a cell tissue structure according to claim 2, wherein the average value of the diameter of all cells, calculated by a weighted average of the number of living cells and the number of dead cells contained in the cells, is used.

4. A method for producing a cell tissue structure according to claim 1, wherein the step of counting the cell diameters is to determine the median diameter of the cells contained in the cell suspension.

5. The method for producing a cell tissue structure according to claim 1, wherein, in the step of counting the cell diameters, cells having a diameter exceeding the maximum diameter and cells having a diameter less than the minimum diameter are excluded from the counting of the cell diameters in the counting step.

6. A method for producing a cell tissue structure according to claim 1 or 2, wherein the cell volume fraction of the cells contained in the cell suspension is 5% or more and 35.7% or less.

7. The cell number concentration in the aforementioned cell suspension is 1.5 × 10⁻⁶. ７ cells / mL or more 1×10 １０ A method for producing a cell tissue structure according to claim 1 or 2, wherein the cell density is less than or equal to cells / mL.

8. The steps include storing the aforementioned mixture in a coating liquid container having a through hole at the bottom, A method for producing a cell tissue structure according to claim 1 or 2, further comprising the step of transferring the mixed solution to an object to be coated by having a coating needle penetrate the coating solution container that contains the mixed solution.

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