Method for producing cell tissue and culture apparatus

Abstract

To provide a cell tissue manufacturing method and culture apparatus with high manufacturing efficiency. [Solution] The method includes the steps of: linearly printing multiple cells between a first substrate and a second substrate such that both ends of the cells are supported by the first substrate and the second substrate, respectively; culturing the printed multiple cells to obtain multiple cell tissues T; filling the space between the first substrate and the second substrate with a liquid retaining material; curing the filled retaining material; separating each of the multiple cell tissues T from at least one of the substrates; and dividing the retaining material along the direction of extension of the multiple cell tissues T held by the cured retaining material to obtain multiple blocks 50C. The step of obtaining multiple blocks 50C includes housing the cured retaining material in a dividing housing 30 having multiple blades 31.

Description

Technical Field

[0001] The present disclosure relates to a method for manufacturing cell tissue and a culture device.

Background Art

[0002] Techniques for constructing cell tissue by bioprinting have been studied. Research is underway to construct cell tissue by bioprinting and use the obtained cell tissue as edible steak meat or processed meat.

[0003] Non-Patent Document 1 discloses a tendon-gel integrated bioprinting and a technique for constructing a tissue such as artificial steak by assembling fibrous cell tissue created by tendon-gel integrated bioprinting in vitro. The tendon-gel integrated bioprinting disclosed in Non-Patent Document 1 is a method in which cells are printed on a gel mimicking a tendon and cultured in a state where both ends of the cells are joined to a support mimicking a tendon.

[0004] In order to realize such a culture method, Patent Document 1 discloses a method of linearly arranging muscle cells in a state where both ends of the cells are joined to a support and culturing the muscle cells in this state.

[0005] Further, Patent Document 2 discloses a method of linearly printing bioink containing cells in a supporting bath, dissolving the gel in the supporting bath after printing, removing the solution in the supporting bath, adding a culture solution, and culturing the cells.

Prior Art Documents

Patent Documents

[0006]

Patent Document 1

Patent Document 2

Non-Patent Documents

[0007] [Non-Patent Document 1] Michiya Matsusaki et al., “Engineered whole cut meat-like tissue by the assembly of cell fibers using tendon-gel integrated bioprinting”, Nature Communications, 12,5059(2021) [Overview of the Initiative] [Problems that the invention aims to solve]

[0008] By culturing cells with both ends supported by a substrate, it is possible to culture the cells while generating tensile tension between the substrate and the cells. This tensile tension facilitates the formation of muscle fibers, muscle tissue, or sarcomere structures, resulting in a cell tissue that more closely resembles animal muscle.

[0009] Thus, while it is preferable to culture cells with both ends supported by a substrate, the substrate must be removed when harvesting the cell tissue obtained after culture. However, cell tissue is very fragile. Therefore, there was a problem in that the cell tissue was damaged when the substrate was removed, reducing the efficiency of production.

[0010] One objective of this disclosure is to provide a method for producing cell tissue and a culture apparatus that prevents damage to cell tissue and improves production efficiency. [Means for solving the problem]

[0011] The present disclosure is a method for producing a plurality of cell tissues. The method for producing a cell tissue includes the steps of: linearly printing a plurality of cells between a first substrate and a second substrate such that both ends of the cells are supported by the first substrate and the second substrate, respectively; culturing the printed plurality of cells to obtain a plurality of cell tissues; filling the space between the first substrate and the second substrate with a soluble liquid retaining material that hardens under predetermined conditions; hardening the filled retaining material; cutting the hardened retaining material from at least one of the first substrate and the second substrate to separate each of the plurality of cell tissues from at least one of the first substrate and the second substrate; and dividing the hardened retaining material along the extending direction of the plurality of cell tissues held by the retaining material to obtain a plurality of blocks. The step of obtaining a plurality of blocks includes placing the hardened retaining material in a dividing housing having a plurality of blades for dividing and housing the hardened retaining material.

[0012] The culture apparatus of this disclosure is a bioprinting culture apparatus for culturing multiple cells separately to obtain multiple cell tissues. The culture apparatus includes a culture housing provided in the order of a first chamber on which a first substrate for supporting multiple cells is arranged, a containment chamber configured to contain a culture medium, and a second chamber on which a second substrate for supporting multiple cells is arranged; a replacement means for replacing the solution contained in the containment chamber with a soluble liquid retaining material that hardens under predetermined conditions; and a separation means. The multiple cells are printed linearly from the first chamber to the second chamber. The separation means separates each of the multiple cell tissues contained in the containment chamber from at least one of the substrates, the first substrate located in the first chamber and the second substrate located in the second chamber. The culture apparatus further includes a division means for dividing the retaining material along the extending direction of the multiple cell tissues held by the hardened retaining material to obtain multiple blocks. [Effects of the Invention]

[0013] According to the present disclosure, the cured holding material can separate the cell tissue from the substrate while holding each of the cell tissues, prevent damage to the cell tissue, and improve the recovery efficiency of the cell tissue.

Brief Description of the Drawings

[0014] [Figure 1] It is a schematic diagram of a manufacturing method showing up to the culturing step. [Figure 2] It is a schematic diagram of a manufacturing method showing steps after the culturing step. [Figure 3] It is a diagram schematically showing the structure of the culturing device according to Embodiment 1. [Figure 4] It is a view of the wall surface in plan view from the normal direction of the flat surface. [Figure 5] It is a diagram showing the printing step and the separation step using the culturing device. [Figure 6] It is a view of the flat surface of the wall surface according to a modification example in plan view. [Figure 7] It is a diagram schematically showing the structure of the culturing housing according to a modification example. [Figure 8] It is a schematic diagram of the manufacturing method according to Embodiment 2. [Figure 9] It is a diagram showing the holding material after the separation step according to Embodiment 3. [Figure 10] It is a diagram showing the holding material after the separation step according to Embodiment 3. [Figure 11] It is a diagram showing the division step according to Embodiment 3. [Figure 12] It is a diagram showing the division step according to Embodiment 3. [Figure 13] It is a diagram showing the dissolution step according to Embodiment 3. [Figure 14] It is a schematic diagram of the manufacturing method according to Embodiment 4. [Figure 15] It is a diagram showing the dissolution step according to a modification example of Embodiment 4. [Figure 16] It is a diagram showing the dissolution step according to a modification example of Embodiment 4. [Figure 17]This figure shows the dissolution process according to a modified example of Embodiment 4. [Modes for carrying out the invention]

[0015] The embodiments of this disclosure will be described in detail below with reference to the drawings. In the drawings, the same or corresponding parts are denoted by the same reference numerals, and their descriptions will not be repeated.

[0016] [Embodiment 1] [Methods for producing cell tissue] The method for producing cell tissue will be described with reference to Figures 1 and 2. Figure 1 is a schematic diagram of the production method up to the culture step. Figure 2 is a schematic diagram of the production method showing steps after the culture step.

[0017] Cellular tissue is obtained by culturing cells printed in a linear fashion. The cells are not particularly limited as long as they form fibrous cellular tissue when printed in a linear fashion and cultured. The cells may be, for example, animal-derived cells, human-derived cells, or cells from animals other than humans. The cells used in this embodiment are, for example, skeletal muscle cells. Skeletal muscle cells may be muscle-derived cells or stem cell-derived cells. Another example of cells used in this embodiment is adipocytes. Yet another example is cells that make up blood vessels. Cellular tissue may be, for example, fibrous muscle, fibrous fat, or fibrous blood vessels.

[0018] Referring to Figures 1 and 2, the method for producing cell tissue according to this embodiment includes a substrate / support material placement step S1, a printing step S2, a support step S3, a first replacement step S4, a culture step S5, a second replacement step S6, a curing step S7, a separation step S8, and a dissolution step S9.

[0019] (Substrate and support material placement process S1) Substrate 1, support material 3, and substrate 2 are arranged in the culture device in that order. That is, support material 3 is positioned so that it is sandwiched between substrate 1 and substrate 2.

[0020] Substrates 1 and 2 only need to bind to and support the cells printed in the printing process S2, and are composed of materials appropriately selected by those skilled in the art. The shape of substrates 1 and 2 may be solid or liquid. The solid state includes a gel state. The physical properties of substrates 1 and 2 are not particularly limited; for example, substrates 1 and 2 may have properties that change shape under predetermined conditions, or properties that do not change shape. Predetermined conditions may be, for example, temperature, pressure, electrical stimulation, pH, etc. Substrates 1 and 2 may be, for example, collagen or collagen nanofibers. The type of collagen is not particularly limited. For example, substrates 1 and 2 may be composed of multiple types of collagen with different types. Furthermore, substrates 1 and 2 may have different compositions from each other. In this embodiment, substrates 1 and 2 will be described as collagen nanofibers.

[0021] Support material 3 is composed of a soluble material. For example, support material 3 is a solution obtained by dissolving a polymeric substance such as gelatin, agar, or gellan gum in an aqueous solvent. In this case, in the base material / support material arrangement step S1, support material 3 may be in gel or sol form. Support material 3 may have thixotropy, meaning that its viscosity decreases and becomes liquid when force is applied, and its viscosity gradually recovers when the force is removed. Support material 3 may also be a mixed solution prepared by, for example, pulverizing a gelled sample and dispersing it in a solvent such as a liquid culture medium. A gelled sample is prepared, for example, by dissolving a polymeric substance such as gelatin, agar, or gellan gum in an aqueous solvent and allowing it to gel. In the base material / support material arrangement step S1, support material 3 will be described as a mixed solution obtained by pulverizing a gelled sample in a gelatin solution and dispersing it in a liquid culture medium.

[0022] In this embodiment, as an example, after placing a liquid substrate 1, the substrate 1 is partially hardened to increase its viscosity, and then a support material 3, which is composed of a mixed solution in which the gelled sample is dispersed in a solvent, is placed on top of the substrate 1. Subsequently, a liquid substrate 2 is placed on top of the support material 3. The method of placing each sample is not particularly limited.

[0023] (Printing process S2) Printing process S2 is a process of printing cells C in a linear fashion. At this time, cells C are printed such that one end of cell C is placed on substrate 1 and the other end is placed on substrate 2. For example, cells C are printed by the following method: A syringe 102 with a nozzle 103 attached to its tip is filled with bio-ink containing cells C. The nozzle 103 is inserted from substrate 2 toward substrate 1 so that the tip of the nozzle 103 is located inside substrate 1. By moving the tip of the nozzle 103 along the direction from substrate 1 toward substrate 2 and ejecting bio-ink from the nozzle 103, cells C are printed in a linear fashion from substrate 1 toward substrate 2.

[0024] Cell printing is performed within the support material 3. If the support material 3 is thixotropic, its viscosity decreases during printing due to the stress generated by the movement of the nozzle 103 and the ejection of the bio-ink, while its viscosity recovers after printing. Because the viscosity decreases during printing, the nozzle can be easily moved and the bio-ink can be easily ejected. On the other hand, because the viscosity recovers and increases after printing, the shape of the printed cells C is maintained by the support material 3 and the cells C are protected. If the support material 3 is a solution of a polymer substance dissolved in an aqueous solvent, the support material 3 may be in sol form for printing, and then made into a gel form after printing.

[0025] The printing process S2 may be performed automatically or semi-automatically using a known three-dimensional bioprinting device. Alternatively, multiple cells may be printed at once using a multi-nozzle dispenser with multiple nozzles.

[0026] The number of linear cells C printed in the printing process S2 is not limited to one or more, but can be two or more, ten or more, 100 or more, 1000 or more, or 10000 or more, and can be appropriately selected depending on the final product to be obtained and the size of the culture apparatus.

[0027] The diameter of the lines formed by cells C when printed is not particularly limited and can be appropriately selected depending on the type of cells C, the viscosity of the bio-ink, the cell tissue to be obtained, the diameter of the available nozzle 103, etc. For example, the diameter may be 100 μm or less, 100 μm or more, 1 mm or less, 1 mm or more, 10 mm or less, 10 mm or more, 100 mm or less, or 100 mm or more.

[0028] In this embodiment, all of the multiple cells C only need to be printed linearly from substrate 1 to substrate 2, and the extension directions of the multiple cells C may or may not be parallel to each other. In this embodiment, each of the multiple cells C is printed such that its extension direction is parallel to each other.

[0029] (Support process S3) Support step S3 is a step in which substrates 1 and 2 are cured to form substrates 1A and 2A, and both ends of cell C are supported by substrates 1A and 2A. In Figures 1 and 2, to make it easier to understand that the shape of substrates 1 and 2 has changed from liquid to solid, the solid substrates are represented as substrates 1A and 2A, and the liquid substrates are represented as substrates 1 and 2. Note that "solid" includes gel-like states, and curing substrates 1 and 2 means altering the substrates 1 and 2 to a shape suitable for supporting the ends of cell C.

[0030] The base materials 1A and 2A may have reversible properties that allow them to change from a solid state to a liquid state, or they may have irreversible properties that prevent them from changing from a solid state to a liquid state.

[0031] If the shape of the substrates 1 and 2 gradually changes from liquid to solid, the printing process S2 and the support process S3 may be performed in parallel. That is, the substrates 1 and 2 may be hardened while the cells C are being printed. By performing the printing process S2 and the support process S3, at least one cell C is printed linearly such that both ends are supported by the substrates 1 and 2.

[0032] Alternatively, the solid (gel) in the support material 3 may be transferred to a liquid (sol) state while the substrates 1 and 2 are curing. For example, if the substrates 1 and 2 do not dissolve under conditions in which the solid (gel) in the support material 3 is transferred to a liquid (sol), the culture apparatus can be controlled to satisfy both the conditions for dissolving the solid (gel) and the conditions for curing the substrates 1 and 2, thereby curing the substrates 1 and 2 while transferring the solid (gel) in the support material 3 to a liquid (sol). By transferring the solid (gel) in the support material 3 to a liquid (sol), the support material 3 can be easily replaced with the culture medium in the next first replacement step S4. In Figure 1, the support material containing the solid (gel) is represented as support material 3, and the support material after the transfer to a liquid (sol) state is represented as support material 3A, with the symbols reversed.

[0033] In this embodiment, the substrates 1 and 2 are collagen nanofibers, and the support material 3 is a mixed solution obtained by pulverizing a gelatin solution and dispersing it in a liquid culture medium. By maintaining the temperature inside the culture device below the temperature at which collagen does not denature, and above the temperature at which the gel in the support material 3 becomes a sol, the substrates 1 and 2 can be hardened while the gel in the support material 3 can be transferred to a sol. For example, the temperature inside the culture device can be maintained at around 40 degrees Celsius or less, 25 degrees Celsius or more, 35 degrees Celsius or more, or 37 degrees Celsius or more. When the temperature inside the culture device is maintained within such a temperature range, the substrates 1 and 2 harden while the gelatin in the support material 3 dissolves. It is preferable that the temperature inside the culture device be maintained at 30 to 40 degrees Celsius, and more specifically at 37 degrees Celsius, taking into consideration the effect on the growth of cell C.

[0034] (1st substitution step S4) The first replacement step S4 is a step of replacing the support material 3A with culture medium 4. For example, culture medium 4 is inserted between substrates 1A and 2A from syringe 104, and the sample (support material 3A) between substrates 1 and 2 is discharged from waste port 105. At this time, by transferring the solid (gel) in the support material 3 to a liquid (sol) in the support material 3 in the support step S3 and transforming it into support material 3A, the support material 3A can be easily removed.

[0035] The culture medium 4 is appropriately selected by those skilled in the art depending on the cell type and other factors. For example, the culture medium 4 may contain differentiation-inducing factors for inducing cell differentiation.

[0036] Although the support process S3 and the process of dissolving the support material 3A are described as being performed in parallel, they may be performed independently. For example, the process of dissolving the support material 3A may be performed after the support process S3, and then the first replacement process S4 may be performed.

[0037] (Culture step S5) The culture step S5 is a step in which cells C are cultured in culture medium 4 to obtain cell tissue T. The culture conditions are appropriately designed by those skilled in the art according to the cell type and the type of cell tissue to be obtained. In addition, the first replacement step S4 may be performed during the culture step S5 to replace the culture medium.

[0038] (Second substitution step S6) Referring to Figure 2, the second replacement step S6 is a step in which the culture medium 4 is replaced with the retaining material 5, and the retaining material 5, which hardens under predetermined conditions, is filled between the base material 1 and the base material 2. For example, the retaining material 5 is inserted between the base materials 1A and 2A from the syringe 106, and the culture medium 4 is discharged from the waste port 107.

[0039] The retaining material 5 is composed of a soluble material and is handled in a dissolved liquid state in the second substitution step S6. The retaining material 5 is a polymeric substance such as gelatin, agar, or gellan gum dissolved in an aqueous solvent. In this embodiment, the retaining material 5 is gelatin dissolved in an aqueous solvent. It is preferable that the retaining material 5 is firm enough to hold the cell tissue T when cured. For example, the retaining material 5 is a gelatin solution in which the gelatin content is adjusted to the range of 35 to 45 mg / mL when using gelatin with a gel strength of 300 g, and more specifically, a gelatin solution in which the gelatin content is adjusted to 40 mg / mL.

[0040] Both the support material 3 and the retaining material 5 are similar in that they are samples containing polymeric substances. In this embodiment, the support material 3 is a solution in which a sample of a gelled polymeric substance is dispersed in a solvent, while the retaining material 5 is a solution in which the polymeric substance is completely dissolved and formed into a sol.

[0041] Furthermore, the retaining material 5 may be used as the culture medium in the culture step S5. For example, a high-molecular-weight substance such as gelatin may be dissolved in the culture medium 4, and cells C may be cultured using this solution. In this case, it is not necessary to perform the second replacement step S6. That is, the step of filling with the retaining material 5 may be performed before the culture step S5, or in parallel with the culture step S5. Also, although the second replacement step S6 is performed by replacing the culture medium 4 with the retaining material 5, the culture medium 4 may be replaced with the retaining material 5 from a different solution. That is, before the second replacement step S6, a pretreatment step to increase the strength of the cell tissue T may be performed, or a step to wash the cell tissue T may be performed.

[0042] (Curing process S7) The curing process S7 is the process of curing the retaining material 5. In Figure 2, to clearly show that the shape of the retaining material 5 has changed from liquid to solid, the liquid retaining material is referred to as retaining material 5 and the solid retaining material as retaining material 5A. For example, the temperature inside the culture device is kept below 20 degrees Celsius, below 10 degrees Celsius, or below 5 degrees Celsius. The curing conditions differ depending on the type of retaining material 5, and are therefore designed according to the type.

[0043] (Separation process S8) The separation step S8 is a step in which the hardened retaining material 5A is separated from the hardened substrates 1A and 2A, thereby separating the substrates 1A and 2A from the cell tissue T. At this time, the cell tissue T is separated from the substrates 1A and 2A while being protected by the retaining material 5A. Therefore, less tensile force is applied to the cell tissue T during separation, and damage to the cell tissue T can be prevented. This makes it possible to increase the recovery rate of the cell tissue T.

[0044] In this embodiment, the separation step S8 may be performed by removing the entire block consisting of the substrates 1A, 2A and the retaining material 5A from the culture device 100. Even during removal, the cell tissue T is protected by the retaining material 5A, which prevents damage to the cell tissue T and increases the recovery rate of the cell tissue T.

[0045] (Dissolution step S9) The dissolution step S9 dissolves the retaining material 5A and recovers the cell tissue T. The dissolution conditions differ depending on the type of retaining material 5A, and are designed accordingly. In one embodiment, a retaining material dissolving solution is used in the dissolution step S9 to dissolve the retaining material 5A. The retaining material dissolving solution is appropriately selected based on the dissolution conditions of the retaining material 5A. Examples of retaining material dissolving solutions include water at a predetermined temperature and a solution with a predetermined pH. More specifically, for example, if the retaining material 5A dissolves due to temperature, the retaining material 5A containing the cell tissue T is dissolved by applying water at a predetermined temperature to the retaining material 5A and recovering the cell tissue T. Alternatively, if the retaining material 5A dissolves due to pH, the retaining material 5A can be dissolved and the cell tissue T recovered by applying a solution with a predetermined pH (e.g., a buffer solution) to the retaining material 5A.

[0046] In one embodiment, when gelatin is used as the material for retaining material 5A, water at a temperature of less than 40 degrees Celsius, 25 degrees Celsius or higher, 35 degrees Celsius or higher, or 37 degrees Celsius or higher (hereinafter referred to as "warm water") is applied to retaining material 5A. In particular, since heat at around 40 degrees Celsius or higher may cause denaturation of cell tissue T (i.e., cell tissue that makes up fibrous muscle, fat, and blood vessels), it is preferable to dissolve the gelatin in water at 37 degrees Celsius. When gellan gum is used as the material for retaining material 5A, a cationic solution such as Tris-HCl buffer, Tris-maleate buffer, or bis-Tris-buffer is applied to retaining material 5A. When agar is used as the material for retaining material 5A, water at 80 degrees Celsius or higher is applied to retaining material 5A.

[0047] Furthermore, considering the impact on cell tissue T, it is preferable that the retaining material 5A is composed of a material that dissolves at a temperature below 40 degrees Celsius or a material that dissolves at a pH of 6.8 to 7.2. However, if the retaining material 5A that has dissolved can be separated from the cell tissue T simultaneously with the dissolution of the retaining material 5, the impact on the cell tissue T can be reduced. Therefore, the retaining material 5 is not limited to dissolving under the above conditions, but may also dissolve under conditions that affect the cell tissue T.

[0048] In the dissolution step S9, instead of pouring the retaining material dissolving solution onto the retaining material 5A containing the cell tissue T, the retaining material 5A containing the cell tissue T may be immersed in a bath containing the retaining material dissolving solution. The "applying the retaining material dissolving solution" device and the "bath containing the retaining material dissolving solution" each correspond to a "dissolution means" for dissolving the retaining material 5A containing the cell tissue T. The "applying the retaining material dissolving solution" device is, for example, a container containing the retaining material dissolving solution, a liquid operation unit for applying the retaining material dissolving solution in the container to the retaining material 5A, and a controller for controlling the liquid operation unit.

[0049] Furthermore, in this embodiment, multiple cells C are printed in the printing process S2, and multiple cell tissues T are obtained. Each of the multiple cells C is printed linearly from substrate 1 toward substrate 2. Therefore, each of the multiple cell tissues T is held in the retaining material 5A with its extension direction aligned.

[0050] Therefore, as shown in Figure 2, when dissolving the retaining material 5A, it is preferable to install and dissolve the retaining material 5A so that the surface on which the retaining material 5A is installed is aligned with the direction of extension of the cell tissue T. This makes it possible to recover multiple cell tissues T with their extension directions aligned. As a result, in the process of assembling the recovered cell tissues T, the step of aligning the fiber direction of the cell tissues T becomes unnecessary.

[0051] Furthermore, since fibrous cell tissue is typically thin (for example, about 1 mm in diameter) and soft, when immersing the retaining material 5A in a bath containing the retaining material dissolving solution, it is preferable to immerse it in a retaining material dissolving solution without flow so that the cell tissue T is not carried away by convection or agitation and the direction of extension of the cell tissue T is not disrupted.

[0052] In this embodiment, both substrates 1A and 2A are separated from the cell tissue T in the separation step S8. Alternatively, at least one of the substrates 1A and 2A may be separated in the separation step S8. If a material that does not dissolve in the dissolution step S9 is used as the substrate, one end of the cell tissue T is retained even after the dissolution step S9, which may make it easier to handle multiple cell tissues T together.

[0053] [Culture apparatus for implementing the separation process] Figure 3 is a schematic diagram showing the structure of the culture apparatus according to Embodiment 1. The culture apparatus 100 is a bioprinting culture apparatus for culturing cells to obtain cell tissue. The culture apparatus 100 comprises a culture housing 10, a replacement means 20, and a detachment means 160.

[0054] The culture enclosure 10 is provided with a first chamber 110 in which the substrate 1 is placed, a storage chamber 120 configured to accommodate the culture medium 4, and a second chamber 130 in which the substrate 2 is placed, in that order.

[0055] The first chamber 110 and the second chamber 130 are configured to hold substrates. The culture apparatus 100 may be provided with substrates already placed in each of the first chamber 110 and the second chamber 130, or it may be configured to allow substrates to be inserted into each of the first chamber 110 and the second chamber 130 later.

[0056] The replacement means 20 replaces the sample (solution) filling the containment chamber 120 with another sample (solution). Specifically, the replacement means 20 includes an inlet 22 configured to accept a tube to which a tube pump is attached or a syringe, and an outlet 24 for discharging the sample from the containment chamber 120 to the outside of the culture device 100. The inlet 22 and the outlet 24 are each connected to the containment chamber 120.

[0057] For example, in the first replacement step S4, the replacement means 20 replaces the sample filling the containment chamber 120 from the support material 3A to the culture medium 4. Also, in the second replacement step S6, after culturing cells C in the culture step S5 to obtain cell tissue T, the replacement means 20 replaces the sample filling the containment chamber 120 from the culture medium 4 to the retaining material 5.

[0058] The separation means 160 is a device for separating the cell tissue T obtained by culturing in the containment chamber 120 from the substrate. The separation means 160 includes a first wall surface 161 and a second wall surface 162. The first wall surface 161 is a wall separating the first chamber 110 and the containment chamber 120 and is located inside the culture housing 10. The second wall surface 162 is a wall separating the containment chamber 120 and the second chamber 130 and is located inside the culture housing 10. The first wall surface 161 is located inside the culture housing 10 such that its flat surface faces the first chamber 110. The second wall surface 162 is located inside the culture housing 10 such that its flat surface faces the second chamber 130.

[0059] Both the first wall surface 161 and the second wall surface 162 are configured to slide. More specifically, the first wall surface 161 is configured to slide along its flat surface. The second wall surface 162 is configured to slide along its flat surface. In Figure 3, the X-axis direction is the sliding direction, the Z-axis direction is the normal direction to the flat surface, and the axis perpendicular to the X and Z axes is the Y-axis. In this embodiment, the first wall surface 161 and the second wall surface 162 are arranged parallel to each other within the culture enclosure 10 and share a common sliding direction. However, the sliding direction of the first wall surface 161 and the sliding direction of the second wall surface 162 may be different.

[0060] When substrates are placed in the first chamber 110 and the second chamber 130, respectively, the first wall surface 161 can be said to be positioned within the culture apparatus 100 so that its flat surface faces the substrate. Similarly, the second wall surface 162 can be said to be positioned within the culture apparatus 100 so that its flat surface faces the substrate.

[0061] Figure 4 is a plan view of the wall surface from the direction normal to the flat surface. Hereafter, the first wall surface 161 and the second wall surface 162 will be collectively referred to as "wall surface 16". An opening 60 is formed in wall surface 16. The plan view shown in Figure 4 is obtained by viewing the flat surface of wall surface 16 from the direction normal to the flat surface of wall surface 16 (Z-axis direction).

[0062] At least one opening 60 is formed in the wall surface 16. The number of openings 60 is not particularly limited and may be 2 or more, 10 or more, 100 or more, 1000 or more, or 10000 or more. Because openings 60 are formed in the first wall surface 161 and the second wall surface 162, the printing process S2 can be carried out without removing the first wall surface 161 and the second wall surface 162.

[0063] The first wall surface 161 and the second wall surface 162 may be plate-like members without an opening 60. In this case, after placing the substrates 1, 2 and the support material 3 in the culture device 100, the first wall surface 161 and the second wall surface 162 are slid to eliminate the gap between the containment chamber 120 and the first chamber 110 and the second chamber 130, and the printing process is performed. Subsequently, in the separation process, the first wall surface 161 and the second wall surface 162 may be slid between the containment chamber 120 and the first chamber 110 and the second chamber 130 to separate the cell tissue T from the substrates 1, 2.

[0064] The size of the opening 60 should be such that it can pass through a nozzle for ejecting a bioink containing cells. If the opening 60 is circular, the diameter of the opening 60 may be 100 μm or less, 100 μm or more, 1 mm or less, 1 mm or more, 10 mm or more, 100 mm or less, or 100 mm or more.

[0065] It is preferable that the first wall surface 161 and the second wall surface 162 are arranged in the culture device 100 such that the opening 60 formed in the first wall surface 161 and the opening 60 formed in the second wall surface 162 are positioned opposite each other.

[0066] As shown in Figure 4, the wall surface 16 may have multiple openings 60 arranged in a two-dimensional pattern. The cells C are printed such that at least one cell C passes through each of the multiple openings 60 arranged in a two-dimensional pattern. In this way, the multiple openings 60 are formed in a two-dimensional pattern on the wall surface 16, making it easier to handle each cell tissue T in the procedure after the cell tissue T is separated from the substrates 1 and 2.

[0067] Multiple openings 60 may be arranged in two directions that intersect each other. The intersection angle can be 30 degrees or less, 30 degrees or more, 45 degrees or less, 45 degrees or more, 90 degrees or less, or 90 degrees or more. In the example shown in Figure 4, multiple openings 60 are arranged in two directions that are orthogonal to each other. Note that multiple openings 60 may also be arranged in a staggered pattern.

[0068] Figure 5 shows the printing and separation processes using the culture device. In the printing process S2A, the cells C can be arranged linearly within the culture device 100 so that both ends of the cells C are located in the first chamber 110 and the second chamber 130 by moving the nozzle 103 for ejecting bio-ink containing cells C as follows. First, the nozzle 103 is positioned so that it passes through the opening 60 of the second wall surface 162 and the opening 60 of the first wall surface 161. Next, while ejecting bio-ink from the nozzle 103, the nozzle 103 is moved so that the tip of the nozzle 103 passes from the opening 60 of the first wall surface 161 to the opening 60 of the second wall surface 162. This allows the cells C to be arranged linearly so that they pass from the opening 60 of the first wall surface 161 to the opening 60 of the second wall surface 162. As a result, the cells C are arranged linearly from the substrate 1 located in the first chamber 110 to the substrate 2 located in the second chamber 130. Subsequently, by curing substrates 1 and 2, both ends of the linear cell C can be supported by substrates 1 and 2.

[0069] Next, in the separation step S8A, the first wall surface 161 and the second wall surface 162 are slid in the X-axis direction along the flat plate surface. As a result, the cell tissue T, which is positioned to pass through the opening 60, is cut by the edge of the opening 60. Consequently, the cell tissue T is separated from the substrates 1A and 2A.

[0070] In this way, the cell tissue T can be easily separated from the substrates 1 and 2 simply by sliding the first wall surface 161 and the second wall surface 162, respectively.

[0071] Note that the arrangement pattern and shape of the openings 60 are not limited to those shown in Figure 4. Figure 6 is a plan view of the flat surface of the wall according to one modified example. The wall surface 16A has an elongated shape in which the length along the X-axis direction (sliding direction) is longer than the length along the Y-axis direction which is perpendicular to the X-axis direction (sliding direction).

[0072] When the opening 60A is elongated, alignment is easier when inserting multiple nozzles, such as a multi-nozzle dispenser, into the culture device 100 at once, compared to when multiple openings 60 are formed in the matrix direction. Also, since the length along the X-axis is shorter than the length along the Y-axis, the distance from the cell tissue T to the edge of the opening 60A can be shortened. This reduces the distance the wall surface 16A slides when separating the substrate from the cell tissue T.

[0073] Furthermore, although the containment chamber 120 is separated into a first chamber 110 and a second chamber 130 by a first wall surface 161 and a second wall surface 162, the containment chamber 120 may also be formed by placing a box inside the culture housing 10. Figure 7 is a schematic diagram showing the structure of a culture housing according to one embodiment. The culture apparatus 100B according to one embodiment may include a box 62 placed inside the culture housing 10 so as to be sandwiched between predetermined spaces (first chamber 110, second chamber 130). The inside of the box 62 corresponds to the containment chamber 120. Openings are formed in the first wall surface 161B which forms the boundary with the first chamber 110 and the second wall surface 162B which forms the boundary with the second chamber 130. The shape of the openings is not particularly limited, and openings of the shape shown in Figure 4 or Figure 6 are formed in the first wall surface 161B and the second wall surface 162B.

[0074] The first wall surface 161B and the second wall surface 162B are each configured to slide along the flat surface of the wall, and at least one of the first wall surface 161B and the second wall surface 162B is configured to open in the Z-axis direction perpendicular to the wall surface.

[0075] By configuring at least one of the first wall surface 161B and the second wall surface 162B to be openable and closable like a lid, the retaining material containing the cell tissue can be easily removed from the culture device 100B after the cell tissue has been separated from the substrate. This makes it easier to handle each cell tissue in the procedure after the cell tissue has been separated from the substrate.

[0076] In this example, the first wall 161 and the second wall 162 separating the chambers within the culture enclosure 10 are designated as the separation means 160, but the configuration of the separation means 160 is not limited to this. For example, the separation means 160 may be realized by providing an opening on the side of the culture enclosure 10 that allows a flat plate to be inserted into the containment chamber 120 in a direction perpendicular to the direction of extension of the cell tissue T (Z-axis direction) (X-axis direction or Y-axis direction). In this case, the first wall 161 and the second wall 162 do not need to be provided.

[0077] [Embodiment 2] In Embodiment 1, after separating the substrates 1A and 2A from the cell tissue T, the retaining material 5A is dissolved in the dissolution step S9. Alternatively, the retaining material 5A may be divided into multiple blocks before dissolving, and then these blocks may be dissolved. Figure 8 is a schematic diagram of the manufacturing method according to Embodiment 2. Note that the manufacturing method according to Embodiment 2 is the same as the manufacturing method shown in Figures 1 and 2 up to the separation step S8; therefore, Figure 8 only shows the steps from the separation step S8 onwards.

[0078] The manufacturing method according to Embodiment 2 differs from the manufacturing method shown in Figures 1 and 2 in that it further includes a dividing step S92 and an alignment step S94, and includes a dissolution step S9A instead of a dissolution step S9.

[0079] (Dividing process S92) The splitting step S92 divides the hardened retaining material 5A along the direction of extension of the cell tissue T to obtain a plurality of blocks 50. Of the plurality of blocks 50, at least one block 50 contains at least one cell tissue T. The number of cell tissues T to be included in each block 50 can be appropriately selected by those skilled in the art and is determined, for example, according to subsequent processing. The number of blocks 50 may be two or more. For example, if the cells C are printed in multiple directions that are orthogonal to each other as shown in Figure 4, the retaining material 5A may be divided into one or more rows.

[0080] In the first embodiment of the division process S92, the retaining material 5A is cut out using a mold having a width corresponding to the width W0 of the desired block 50. In the second embodiment, the retaining material 5A is cut by a cutting device (cutter, metal plate, etc.) at intervals corresponding to the width of the desired block 50. The mold in the first embodiment and the cutting device in the second embodiment correspond to an embodiment of the "division means" that divides the hardened retaining material 5A along the direction of extension of the cell tissue T held in the retaining material 5A to obtain multiple blocks. Furthermore, the width of the mold in the first embodiment and the cutting interval in the second embodiment correspond to an embodiment of the "spacing of the blades that divide the retaining material in the division process" which will be described later.

[0081] By dividing the retaining material 5A, the cell tissue can be handled in units of 50 blocks, and since the cell tissue is protected by the hardened retaining material 5A that makes up the block 50, the cell tissue becomes easier to handle. Also, by dissolving in units of 50 blocks, it is easy to obtain bundles consisting of one or more cell tissues T. Furthermore, when combining multiple types of cell tissues T to create an aggregate that mimics the structure of a specific tissue, combining them in units of 50 blocks is easier than combining them individually.

[0082] (Alignment process S94) The alignment step S94 is a step of aligning and arranging a plurality of blocks 50. The plurality of blocks 50 are arranged so that the direction of extension of the cell tissue T is aligned with each other. At this time, each of the plurality of blocks 50 may be placed on a mesh 200 having a wavy structure. When placing the blocks 50 on a mesh 200 having a wavy structure, the blocks 50 may be placed in the recesses 220 so that the direction of extension of the recesses 220 is aligned with the direction of extension of the cell tissue T contained in the blocks 50. In the example in Figure 8, the plurality of blocks 50 are arranged spaced apart, so the alignment step S94 is also an embodiment of a "separation step" for separating the plurality of blocks. Also, in the example in Figure 8, the equipment for separating the plurality of blocks 50 corresponds to an embodiment of a "separation means". By separating the plurality of blocks 50, the dissolution rate can be increased compared to when they are not separated (details will be described later).

[0083] (Dissolution process S9A) The dissolution step S9A is performed after the alignment step S94. By dissolving the retaining material 5A after the multiple blocks 50 have been arranged so that the extension directions of the cell tissue T are aligned, multiple cell tissues T can be obtained with their extension directions aligned. Therefore, there is no need to align the extension directions of the cell tissue T with tweezers or the like, and the risk of damaging the cell tissue T can be reduced. Note that the dissolution conditions are the same as in Embodiment 1 above, so the explanation is omitted.

[0084] Preferably, when the block 50 is placed on the wavy mesh 200 and then dissolved, the cell tissue T will be collected in the recesses 220, making it easier to obtain bundles of cell tissue T with aligned extension directions.

[0085] [Embodiment 3] In Embodiment 3, the retaining material 5A is housed in a divided housing having multiple blades, and the cell tissue is recovered by dissolving the retaining material 5A within the divided housing.

[0086] The manufacturing method according to Embodiment 3 is the same as the manufacturing method shown in Figures 1 and 2 up to the separation step S8. Figures 9 and 10 show the retaining material 5A after the separation step S8 according to Embodiment 3.

[0087] In Figure 9 and subsequent figures, the extension direction of the cell tissue T held by the retaining material 5A is defined as the Z-axis direction, and the plane perpendicular to the Z-axis direction is defined as the XY plane. In one embodiment, during the dissolution process, the direction that is vertically upward is defined as the positive Y-axis direction, and the direction that is vertically downward is defined as the negative Y-axis direction. In Figure 9 and subsequent figures, the positive X-axis direction may be expressed as right, and the negative X-axis direction as left. Also, the positive Y-axis direction may be expressed as up, and the negative Y-axis direction as down. Furthermore, the positive Z-axis direction may be expressed as front, and the negative Z-axis direction as back. In addition, the length in the X-axis direction may be expressed as width, and the Z-axis direction as depth.

[0088] Figure 9 shows the retaining member 5A as viewed from the Z-axis direction. Figure 10 shows the retaining member 5A as viewed from the X-axis direction.

[0089] In the examples shown in Figures 9 and 10, the cell tissue T in the retaining material 5A is configured to be aligned at equal intervals and parallel to one another. The length of the retaining material 5A along the X-axis is L1, the length along the Y-axis is L2, and the length along the Z-axis is L3. The spacing of the retaining material 5A along the X-axis is L11, and the spacing along the Y-axis is L12.

[0090] In one embodiment of Embodiment 3, the retaining material 5A containing the cell tissue T is prepared as follows. First, a dispensing device is prepared that has six syringes spaced 9 mm apart to match the well arrangement of an 8 × 12 well (9 mm spacing) 96-well plate containing bioink containing cells such as muscle cells. Using this dispensing device, 12 rows are printed with 8 cells C per row, each with a fiber length of 20 mm, with the feed rate in the X and Y directions set at 4.5 mm intervals. Then, the cell tissue T is cultured to obtain the cell tissue T, and the area around the cell tissue T is replaced with gelatin. By cooling and hardening the gelatin to 4 degrees Celsius, the cell tissue T can be fixed in the gelatin. In one embodiment, the size of the hardened gelatin is configured to be 54 mm × 36 mm × 20 mm. In other words, in this embodiment, L1, L2, L3, L11, and L12 in Figures 9 and 10 are 54 mm, 36 mm, 20 mm, 4.5 mm, and 4.5 mm, respectively.

[0091] Figures 11 and 12 show the division process S92C according to Embodiment 3. In the division process S92C, the retaining material 5A is divided to obtain multiple blocks 50C by housing the retaining material 5A in the divided housing 30, as shown in Figures 9 and 10. Figure 11 is a view of the divided housing 30 containing the retaining material 5A from the Z-axis direction, and Figure 12 is a view of the divided housing 30 containing the retaining material 5A from the X-axis direction.

[0092] The divided housing 30 includes a plurality of blades 31 for dividing and housing the hardened retaining material 5A, a wall surface 32, and a bottom surface 33. The divided housing 30 is typically a rectangular parallelepiped housing with an open top. The divided housing 30 may have an upper surface, in which case it is preferable to provide a hole in the upper part of the divided housing 30 for passing the retaining material dissolving liquid 36 through.

[0093] The blade 31 is a partition plate for creating and storing multiple blocks 50C. In the division process S92C, the hardened retaining material 5A is pushed into the division housing 30, thereby dividing the retaining material 5A with the blade 31. As described above, the blade 31 only needs to be able to cut the hardened retaining material 5A, and it is preferable that it be made of a material harder than the hardened retaining material, but it does not necessarily need to have a sharply pointed tip. By using a division housing 30 that includes multiple blades 31 arranged at intervals W1, multiple blocks 50C having a width W1 can be obtained.

[0094] In one embodiment, when the retaining material (e.g., gelatin) is melted by heat during the melting process, it is preferable that the divided housing 30 be formed from a material with high thermal conductivity (e.g., metal).

[0095] Of the multiple blocks 50C, at least one block 50C contains at least one cell tissue T. The number of cell tissues T to be included in each block 50C can be appropriately selected by those skilled in the art and is determined, for example, depending on subsequent processing. The number of blocks 50C may be two or more. For example, if multiple cell tissues T are printed in two mutually orthogonal directions as shown in Figure 9, the divided housing 30 may be configured to divide the retaining material 5A into one or more rows.

[0096] In the examples in Figures 11 and 12, a divided housing 30 is used with blade spacing W1 such that each block 50C contains one row of cell tissue T. In other words, the blade spacing W1 is designed to be the same as, for example, the spacing L11 of the cell tissue T. This results in multiple blocks 50C, each containing one row of cell tissue.

[0097] In one embodiment, the retaining material 5A is pressed into the divided housing 30 parallel to the extension direction (Z-axis direction) of the cell tissue T. In this case, it is preferable that the front or rear wall of the divided housing 30 be configured to open and close as a lid. For example, the lid of the divided housing 30 is configured to be removable. This prevents the Z-axis position of the cell tissue T separated from the retaining material 5A from shifting by opening the lid of the divided housing 30, pressing in the retaining material 5A, and then closing the lid. This also prevents the cell tissue T from overflowing or falling out from the bottom surface 33.

[0098] In one embodiment, the rear wall is configured as a lid, and the thickness of the lid is different from the thickness of the front wall. For example, if the thickness of the lid is greater than that of the front wall, the front portion of each block 50C, where heat is easily conducted, will dissolve faster than the rear portion. This may cause the rear of the cell tissue T to sag, bend, or stick to the blade or wall. Therefore, it is preferable to configure the thickness of the lid to be the same as that of the front wall. Alternatively, as will be described later regarding the recovery housing 40 in Embodiment 4, it is preferable to configure the length of the divided housing 30 in the Z-axis direction to be longer than the length of the block 50C, and to provide a gap between the block 50C and the divided housing 30.

[0099] In other embodiments, the retaining material 5A is pressed parallel to the Y-axis direction of the divided housing 30. Preferably, the lower part of the divided housing 30 is provided with holes for discharging the dissolved retaining material 5A. These holes are configured so as not to allow cell tissue T to pass through. For example, at least one such hole is formed in the lower part of the wall surface 32 and / or the bottom surface 33. More preferably, at least one such hole is formed between adjacent blades. Even more preferably, the bottom surface 33 is configured as a mesh containing a large number of holes. The mesh is, for example, 100 mesh (a mesh in which 100 x 100 strands are woven into a square with sides of 25.4 mm). This allows the dissolved retaining material 5A to pass through the holes in the bottom surface 33, and the cell tissue T separated by the retaining material 5A can be held on the bottom surface 33 parallel to the bottom surface 33. Furthermore, the more numerous and widely distributed the holes are, like in a mesh, the more the dissolved retaining material 5A can pass through a wider area of ​​the bottom surface 33, resulting in faster and more uniform dissolution.

[0100] Figure 13 shows the dissolution process S9C according to Embodiment 3. The dissolution process S9C is performed after the division process S92C. Figure 13(A) shows the divided housing 30, which houses multiple blocks 50C, immersed in the retaining material dissolving solution 36 contained in the bath 35. Figure 13(B) shows the state in which the retaining material 5A of the blocks 50C has dissolved as a result of immersing the divided housing 30 in the retaining material dissolving solution 36. In Figure 13(B), only a part of Figure 13(A) (the part enclosed by the dashed line 37) is shown.

[0101] As shown in Figure 13, by immersing the divided housing 30 in the retaining material dissolving solution 36, the retaining material 5A of the multiple blocks 50C housed in the divided housing 30 dissolves and is discharged to the outside of the divided housing 30. As a result, the cell tissue T is aligned between adjacent blades 31, or between adjacent blades 31 and the wall surface 32. This makes it easy to obtain multiple cell tissues T with their extension directions aligned.

[0102] The dissolution conditions for Embodiment 3 are the same as those for Embodiment 1. Furthermore, it is preferable that the retaining material dissolving solution 36 has a lower specific gravity than the retaining material 5A. This allows the retaining material 5A dissolved in the divided housing 30 to pass through the holes at the bottom of the divided housing 30 by its own weight. Therefore, the rate of dissolution of the block 50C in the divided housing 30 can be accelerated and made uniform.

[0103] Furthermore, it is preferable that the retaining material dissolving solution 36 has a lower specific gravity than the cell tissue T. As a result, the cell tissue T separated from the retaining material 5A in the dividing housing 30 sinks to the bottom surface 33 by its own weight. Therefore, it is possible to prevent the cell tissue T from being parallel to the bottom surface 33 between adjacent blades 31 in the dividing housing 30. The state in which the cell tissue T is not parallel to the bottom surface 33 means, for example, that at least a part of the cell tissue T is bent, twisted, entangled with each other, or sticks to the blades 31 and / or the wall surface 32, and is therefore not parallel to the bottom surface 33.

[0104] In one embodiment, the retaining material 5A is gelatin, the retaining material dissolving solution 36 is hot water, and the bath 35 is a constant temperature water bath. With this configuration, the cell tissue T can be safely recovered without damaging it.

[0105] Furthermore, if the bottom surface 33 of the divided housing 30 is configured to be removable from the wall surface 32, it is easy to recover the cell tissue T on the bottom surface 33 without disturbing its alignment.

[0106] As described above, according to Embodiment 3, the retaining material 5A can be easily divided by housing it in a divided housing 30 having a plurality of blades 31. Then, by simply immersing the divided housing 30 in the retaining material dissolving solution 36, aligned cell tissue T can be easily obtained. Therefore, unlike Embodiment 1, there is no need to align the cell tissue T with tweezers or the like. Also, compared to Embodiment 2, the alignment step S94 in which a plurality of blocks 50 are aligned and placed is omitted, thus saving time. In particular, according to Embodiment 3, in the alignment step S94 of Embodiment 2, there is no possibility that the user may mistakenly place the blocks 50 so that the extending direction of the recess 220 and the extending direction of the cell tissue T contained in the blocks 50 do not align. Therefore, cell tissue T can be recovered with aligned extending directions more easily and accurately than in Embodiments 1 and 2.

[0107] [Embodiment 4] In Embodiment 4, the retaining material 5A is divided to obtain multiple blocks, and then the multiple blocks are separated before the retaining material 5A is dissolved.

[0108] Figure 14 is a schematic diagram of the manufacturing method according to Embodiment 4. The manufacturing method according to Embodiment 4 is the same as the manufacturing method of Embodiment 3 up to the division process S92C.

[0109] Referring to Figure 14, the retaining material 5A containing the cell tissue T after the separation step S8 is divided into multiple blocks 50C in the division step S92C.

[0110] The separation step S94D in Embodiment 4 is a step of separating a plurality of blocks 50C. The separation step S94D is performed after the division step S92C and before the dissolution step S9D.

[0111] The separation step S94D separates the multiple blocks 50C using a predetermined separation means. In the example in Figure 14, the separation step S94D separates the multiple blocks 50C using the recovery housing 40.

[0112] The collection housing 40 includes a partition 41, a wall surface 42, a bottom surface 43, and a top surface 44. Preferably, the upper and lower parts of the collection housing 40 are provided with holes for the retention material dissolving solution 36 and the dissolved retention material 5A to pass through. In one embodiment, at least a portion of the bottom surface 43 and the top surface 44 is made of mesh. In another embodiment, the holes in the upper part of the collection housing 40 may be gaps provided between at least one wall surface 42 and the top surface 44. Alternatively, the upper part of the collection housing 40 may be left open.

[0113] The recovery enclosure 40 has multiple rooms 45. The multiple rooms 45 include a room 45A surrounded by a bottom surface 43 and adjacent partitions 41 and a wall surface 42, and a room 45B surrounded by the bottom surface 43 and two adjacent partitions 41. Each of the multiple rooms 45 has a width W2 that is wider than the spacing W1 between the blades 31 that divide the retaining material 5A in the dividing process S92C to obtain multiple blocks 50C. In room 45A, the width W2 is the spacing between adjacent partitions 41 and the wall surface 42, and in room 45B, it is the spacing between two adjacent partitions 41. Thus, the width W2 of the room 45 is wider than the width W1 of the block 50C to be inserted. As a result, when a block 50C is inserted into a room 45, a gap 46 is created between the block 50C and the partition 41 or wall surface 42.

[0114] In one embodiment, a block 50C made of gelatin containing cell tissue T is formed with a width of 4.5 mm and a depth of 20 mm, and a collection housing 40 is used with partitions 28 mm deep spaced at 6.5 mm intervals. This allows for a gap of 1 mm on the left and right and 4 mm in the front and back between the block 50C and the wall surface 42 of the collection housing 40.

[0115] Furthermore, in the dissolution process S9D described later, when the retaining material (e.g., gelatin) is dissolved by heat, it is preferable that the recovery housing 40 be made of a material with high thermal conductivity (e.g., metal).

[0116] In the separation process S94D shown in Figure 14, the multiple blocks 50C are separated by placing each of them into a recovery housing 40 having multiple chambers 45 with a width W2. In one embodiment, the multiple blocks 50C are inserted into the recovery housing 40 parallel to the direction of extension (Z-axis direction) of the cell tissue T. In this case, it is preferable that the front or rear wall of the recovery housing 40 be configured to open and close as a lid. As an example, the lid of the recovery housing 40 is configured to be removable. This prevents the Z-axis position of the cell tissue T separated from the retaining material 5A from shifting by opening the lid of the recovery housing 40, inserting the blocks 50C, and then closing the lid. This also prevents the cell tissue T from overflowing or falling out from the bottom surface 43.

[0117] In one embodiment, each block 50C is inserted into each chamber 45 using a push-in section 49 having approximately the same width as each block 50C. The push-in section 49 may be controlled by a controller (not shown) or moved manually by the user. As described above, by using the retrieval housing 40, each of the multiple blocks 50C can be easily separated. The retrieval housing 40 and the push-in section 49 correspond to one embodiment of the "separation means".

[0118] In the dissolution process S9D shown in Figure 14, the retaining material 5A is dissolved by immersing the recovery housing 40 containing the block 50C in the retaining material dissolving solution 36, thereby allowing for easy recovery of the cell tissue T. In the dissolution process S9D shown in Figure 14, the retaining material dissolving solution 36 enters the gap 46 between each block 50 and the partition 41 or wall surface 42, causing the retaining material dissolving solution 36 to also come into contact with the side surface of the block 50 (the surface facing the partition 41 or wall surface 42). The retaining material 5A dissolves faster in the parts that come into contact with the retaining material dissolving solution 36 than in the parts that do not come into contact with the retaining material dissolving solution 36. The parts that do not come into contact with the retaining material dissolving solution 36 include, for example, the parts of the retaining material 5A that come into contact with the partition 41 or wall surface 42 and the internal parts of the retaining material 5A. As described above, compared to Embodiment 3, in which each block 50C is not separated (there is no gap between each block 50 and the partition 41 or wall surface 42), the retaining material 5A of each of the multiple blocks 50 can be dissolved quickly and uniformly. In particular, according to Embodiment 4, even the block 50C located in the central part of the divided housing 30, which is thought to be relatively difficult to dissolve and therefore takes a relatively long time to dissolve in Embodiment 3, can be dissolved quickly. As described above, by separating the multiple blocks 50C, the cell tissue T can be recovered more quickly, and bundles of cell tissue T with a more aligned direction of extension can be obtained.

[0119] In this specification, "multiple blocks are not spaced apart" includes not only cases where multiple blocks are in contact with each other and lined up without gaps, but also cases, as in Embodiment 3, where blocks are lined up without gaps via a blade. More specifically, "multiple blocks are not spaced apart" refers to cases where the divided cross-sections of the hardened retaining material 5A are not spaced apart.

[0120] In the separation step S94D, the manner in which multiple blocks are separated is not limited to the example in Figure 8. For example, in the division step S92C, the width of each block may be reduced by cutting off the portion of each block that does not contain the cell tissue T, thereby separating the multiple blocks. In the embodiment 2 described above (Figure 8), the separation step is shown in which the blocks separated in the division step S92 are placed at separated positions.

[0121] As described above, according to the cell tissue manufacturing method of Embodiment 4, by separating the multiple blocks 50C, the multiple blocks 50C can be dissolved quickly and uniformly, and therefore, the cell tissue T can be recovered quickly and in an aligned state. In particular, according to the example in Figure 14, by using the recovery housing 40, the multiple blocks 50C can be separated easily and reliably.

[0122] In the above description, we mainly explained a configuration in which each block 50C is spaced apart along the X-axis. However, if the wall surface 32 in the Z-axis direction is also spaced apart from each block 50C, the dissolution rate of each block 50C can be further increased. In other words, if there is a gap between the front wall surface and the rear wall surface and each block 50C, the dissolution efficiency can be improved. Furthermore, with this configuration, even if the thickness of the lid (for example, the rear wall surface) and the thickness of the wall surface opposite the lid (for example, the front wall surface) are not uniform in the recovery enclosure 40, it becomes possible to make the dissolution rate of the retaining material 5A near the lid and the dissolution rate of the retaining material 5A near the wall surface opposite the lid uniform.

[0123] [Modified form of Embodiment 4] In the modified embodiment of Embodiment 4, the multiple blocks are stored in the recovery enclosure such that none of the multiple blocks are stored in adjacent rooms. Hereafter, the modified embodiment of Embodiment 4 will also be referred to simply as the modified embodiment. The manufacturing method according to the modified embodiment is the same as the manufacturing method shown in Embodiments 3 and 4 up to the division step S92C.

[0124] In the modified example, the separation step S94Da is performed after the splitting step S92C and before the dissolution step S9Da.

[0125] The separation process S94Da involves placing the multiple blocks 50C into the recovery enclosure 40a so that none of the multiple blocks 50C are placed in adjacent rooms.

[0126] The recovery housing 40a corresponds to one embodiment of the "separation means". The recovery housing 40a may be configured in the same way as the recovery housing 40, but in one embodiment, the width W3 of the chamber 45 is configured to be equivalent to the spacing W1 of the blades that divide the retaining material 5A in the division process S92C (details will be described later). The wall surface 42a of the recovery housing 40a includes the wall surface 42Aa on the positive Z-axis side, the wall surface 42Ba on the negative Z-axis side, and the wall surface 42Ca which is horizontal to the YZ plane. In one embodiment, the wall surface 42Aa is configured to be openable and closable as a lid. This makes it possible to prevent the Z-axis position of the cell tissue T separated from the retaining material 5A from shifting by opening the lid, inserting the block 50C, and then closing the lid. This also makes it possible to prevent the cell tissue T from overflowing or falling out from the bottom surface 43a.

[0127] The collection enclosure 40a has multiple rooms 45a. The multiple rooms 45a include a room 45Aa surrounded by a bottom surface 43a and adjacent partitions 41a and a wall surface 42Ca, and a room 45Ba surrounded by the bottom surface 43a and two adjacent partitions 41a.

[0128] The separation process S94Da is carried out in roughly the same manner as the separation process S94D, but differs in that after inserting one block 50C into one room 45a, one or more rooms 45a are skipped and the next block 50C is inserted into the next room 45a. As a result, in the retrieval enclosure 40a, each of the multiple blocks 50C is stored with one or more rooms 45a empty between them. Therefore, the rooms 45a on either side of the room 45a into which each block 50C is inserted become empty rooms into which no blocks 50C have been inserted.

[0129] In one embodiment, the separation step S94Da, as shown in Figures 15 to 17, places each block 50C into a room 45a with one block skipped between them. This allows for the most efficient insertion of blocks 50C while the rooms adjacent to the room into which each block 50C is inserted are empty. In other words, the largest number of blocks 50C can be stored with empty rooms on either side.

[0130] Figures 15 to 17 show the dissolution process S9E according to a modified example. In the dissolution process S9Da shown in Figures 15 to 17, a recovery housing 40a containing multiple blocks 50C inserted with one or more chambers 45a between them is immersed in a retaining material dissolution solution 36. As a result, in one embodiment, even if the width W3 of the chambers is equal to the width W1 of the blocks 50C and there is no gap between the blocks 50C and the partitions 41a or walls 42Ca, heat is transferred to the sides of the blocks 50C via the partitions 41a and walls 42Ca, allowing the heat-melting retaining material 5A to be uniformly dissolved. Furthermore, blocks 50C located closer to the center of the recovery housing 40a can be dissolved at the same speed as blocks 50C located further out of the recovery housing 40a.

[0131] However, in the recovery enclosure 40a according to one embodiment, if the wall surface 42Ca, which requires more strength than the partition 41a, is configured to be thicker, when a block 50C is inserted into the room 45Ba, the dissolution of the retaining material 5A takes longer than when a block 50C is inserted into the room 45Aa. In this case, it is preferable not to insert a block 50C into the room 45Ba.

[0132] In one embodiment, when the dissolution process S9Da according to the modified example was performed using a recovery housing 40a configured similarly to the divided housing 30 of Embodiment 2, the dissolution process, which took approximately 20 minutes in Embodiment 2, was shortened to approximately 6 minutes. The time required for the above dissolution process is the time from when the housing containing multiple blocks 50C is submerged in the retaining material dissolving solution 36 to start dissolution until all the retaining material 5A of the blocks 50C is completely dissolved.

[0133] In the dissolution process S9D according to the aforementioned embodiment 4, in order to separate the blocks 50C, it is necessary to position the blocks 50C in each chamber 45 in a position that leaves a gap between the block 50C and the partition 41. On the other hand, according to the dissolution process S9Da according to the modified example, it is convenient that the blocks 50C can be reliably separated simply by placing one or more blocks 50C in each chamber 45a without any gaps between them. However, in the dissolution process S9D according to embodiment 4, if the blocks 50C are positioned so that there is a gap between them and the partition, the retaining material dissolving liquid 36 can come into contact with the side surface of the block 50C instead of the partition, which is superior in terms of the dissolution speed of the block 50C.

[0134] Furthermore, in the modified recovery enclosure 40a, similar to the recovery enclosure 40 in Embodiment 4, the width of the chamber 45a may be made larger than the width of the block 50C so that there is a gap between the block 50C and the partition 41a. This makes it possible to further increase the dissolution rate of each block 50C.

[0135] Furthermore, in the recovery enclosure 40a, it is preferable to configure it so that the rear wall surface 42Aa and the front wall surface 42Ba are spaced apart from the block 50C. This further increases the dissolution rate of each block 50C, and, as shown in Figure 15, even in embodiments where the thickness of the lid (wall surface 42Aa) and the wall surface opposite the lid (wall surface 42Ba) are not uniform, the dissolution rate of the retaining material 5A near the lid and the dissolution rate of the retaining material 5A near the wall surface opposite the lid can be made uniform.

[0136] As described above, Embodiments 1 to 4 and their modifications provide a method for manufacturing cell tissue and a culture apparatus that prevents damage to cell tissue and improves manufacturing efficiency. In particular, Embodiments 2 to 4 and their modifications allow for the division of block 50C into desired sizes before dissolution, thereby obtaining small, divided cell tissue T with aligned extension directions. For example, dividing block 50C into equal parts yields equally divided cell tissue T. This eliminates the need for the conventional step of using tweezers to align the extension directions of cell tissue T, which has varying extension directions, significantly reducing working time. It also eliminates the need to divide the cell tissue T into desired quantities. Furthermore, Embodiments 3 to 4 and their modifications, compared to Embodiment 2, allow for the more accurate and simpler acquisition of bundles of cell tissue T with aligned extension directions due to the absence of an alignment step. Moreover, Embodiment 4 and its modifications, compared to Embodiment 3, allow for the dissolution of block 50C more uniformly and quickly, thus reducing the time required for the dissolution process.

[0137] [Pattern] Those skilled in the art will understand that the embodiments described above are specific examples of the following embodiments.

[0138] (Section 1) A method for producing cell tissue according to one embodiment is a method for producing a plurality of cell tissues. The method for producing cell tissues includes the steps of: linearly printing a plurality of cells between a first substrate and a second substrate such that both ends of the cells are supported by the first substrate and the second substrate, respectively; culturing the printed plurality of cells to obtain a plurality of cell tissues; filling the space between the first substrate and the second substrate with a soluble liquid retaining material that hardens under predetermined conditions; hardening the filled retaining material; cutting the hardened retaining material from at least one of the first substrate and the second substrate to separate each of the plurality of cell tissues from at least one of the first substrate and the second substrate; and dividing the hardened retaining material along the direction of extension of the plurality of cell tissues held by the retaining material to obtain a plurality of blocks. The step of obtaining a plurality of blocks includes storing the hardened retaining material in a dividing housing having a plurality of blades for dividing and storing the hardened retaining material.

[0139] According to the cell tissue manufacturing method described in paragraph 1, damage to the cell tissue can be prevented and the cell tissue recovery efficiency can be improved by separating the cell tissue from the first and second substrates while each cell tissue is held in place by the hardened retaining material. Furthermore, the cell tissue can be handled in block units, and since the cell tissue is protected by the hardened retaining material that makes up the block, the cell tissue becomes easier to handle. In addition, when combining multiple types of cell tissue to create an aggregate that mimics the structure of a specific tissue, combining them in block units is easier than combining them individually. Moreover, the retaining material can be easily divided by using a divisible enclosure. Therefore, it is possible to prevent damage to the cell tissue and provide a cell tissue manufacturing method with high manufacturing efficiency.

[0140] (Section 2) The method for producing cell tissue described in Section 1 further includes the step of dissolving the retaining material constituting each of the multiple blocks housed in the divided enclosure to recover the multiple cell tissues.

[0141] According to the method for producing cell tissue described in paragraph 2, aligned cell tissue can be easily obtained.

[0142] (Section 3) The method for producing cell tissue described in Section 1 or 2 further includes the step of separating a plurality of blocks.

[0143] According to the method for producing cell tissue described in paragraph 3, cell tissue can be recovered more quickly, and a bundle of cell tissue with a more aligned direction of extension can be obtained.

[0144] (Clause 4) In the method for producing cell tissue described in paragraph 3, the step of separating a plurality of blocks includes the step of placing each of the plurality of blocks in a recovery housing having a plurality of chambers having a width wider than the spacing between the blades that divide the retaining material in the step of obtaining the plurality of blocks.

[0145] According to the method for producing cell tissue described in paragraph 4, multiple blocks can be easily and reliably separated.

[0146] (Clause 5) In the method for producing cell tissue described in paragraph 3 or 4, the step of separating a plurality of blocks includes the step of storing the plurality of blocks in a recovery enclosure having a plurality of compartments such that each of the plurality of blocks is not stored in a compartment adjacent to one another.

[0147] The method for producing cell tissue described in Section 5 is convenient because it allows for reliable separation of blocks simply by placing them in a room with one or more blocks skipped between them.

[0148] (Clause 6) A culture apparatus according to one embodiment is a culture apparatus for bioprinting to obtain multiple cell tissues by culturing multiple cells separately. The culture apparatus includes a culture housing provided in the order of a first chamber on which a first substrate for supporting multiple cells is arranged, a containment chamber configured to contain a culture medium, and a second chamber on which a second substrate for supporting multiple cells is arranged; a replacement means for replacing the solution contained in the containment chamber with a soluble liquid retaining material that hardens under predetermined conditions; and a separation means. Multiple cells are printed linearly from the first chamber to the second chamber. The separation means separates each of the multiple cell tissues contained in the containment chamber from at least one of the first substrate located in the first chamber and the second substrate located in the second chamber. The culture apparatus further includes a division means for dividing the retaining material along the direction of extension of the multiple cell tissues held by the hardened retaining material to obtain multiple blocks.

[0149] According to the culture apparatus described in paragraph 6, a retaining material that hardens under predetermined conditions can be inserted into the containment chamber by a replacement means, allowing the cell tissue to be separated from the substrate while each cell tissue is held in place by the hardened retaining material. This prevents damage to the cell tissue and improves the efficiency of cell tissue recovery. Furthermore, the cell tissue can be handled in block units, and since the cell tissue is protected by the hardened retaining material that makes up the block, the cell tissue becomes easier to handle. In addition, when combining multiple types of cell tissue to create an aggregate that mimics the structure of a specific tissue, combining them in block units is easier than combining them individually. Moreover, the retaining material can be easily divided by using a divisible enclosure. Therefore, it is possible to provide a cell tissue manufacturing apparatus that prevents damage to the cell tissue and improves manufacturing efficiency.

[0150] (Section 7) The culture apparatus described in Section 6 further comprises a lysis means for dissolving the retaining material and recovering multiple cell tissues.

[0151] According to the culture apparatus described in paragraph 7, cell tissue can be recovered by a simple method of dissolving the excised retaining material.

[0152] (Section 8) The culture apparatus described in Section 6 or 7 further comprises means for separating a plurality of blocks.

[0153] According to the culture apparatus described in paragraph 8, cell tissue can be harvested more quickly, and bundles of cell tissue with more aligned extension directions can be obtained.

[0154] The embodiments disclosed herein are intended to be implemented in appropriate combinations, to the extent that they do not contradict the technical invention. Furthermore, 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 description of the embodiments described above, and all modifications within the meaning and scope equivalent to the claims are intended to be included. [Explanation of Symbols]

[0155] 1,1A,2,2A Substrate, 3,3A Support material, 4 Culture medium, 5,5A Retaining material, 10 Culture enclosure, 16,16A Wall surface, 20 Replacement means, 22 Insertion port, 24 Discharge port, 30 Divided enclosure, 31 Blade, 32 Wall surface, 33 Bottom surface, 35 Bath, 36 Retaining material dissolving solution, 37 Part, 40,40a Recovery enclosure, 42,42a,42Aa,42Ba,42Ca Wall surface, 43,43a Bottom surface, 44 Top surface, 45,45a,45A,45Aa,45B,45Ba Chamber, 46 Gap, 49 Push-in part, 50,50C Block, 60,60A Opening, 62 Box, 100,100B Culture device, 102,104,106 Syringe, 103 Nozzle, 105, 107 Discharge port, 110 First chamber, 120 Containment chamber, 130 Second chamber, 160 Separation means, 161, 161B First wall, 162, 162B Second wall, 200 Mesh, 220 Recess, C cell, L11 Spacing, T cell tissue, W0, W1, W2, W3 Spacing (width).

Claims

1. A method for producing multiple cell tissues, The aforementioned manufacturing method is The steps include: printing multiple cells linearly between a first substrate and a second substrate such that both ends of the cells are supported by the first substrate and the second substrate, respectively; The steps include culturing the printed plurality of cells to obtain the plurality of cell tissues, The steps include filling the space between the first substrate and the second substrate with a soluble liquid retaining material that hardens under predetermined conditions, The steps include: curing the filling retaining material, The steps include cutting the cured retaining material from at least one of the first substrate and the second substrate, thereby separating each of the plurality of cell tissues from at least one of the first substrate and the second substrate, The process includes the step of dividing the hardened retaining material along the direction of extension of the plurality of cell tissues held by the retaining material to obtain a plurality of blocks, A method for producing cell tissue, comprising the step of obtaining the plurality of blocks, which includes the step of housing the cured retaining material in a dividing housing having a plurality of blades for dividing and housing the cured retaining material.

2. A method for producing cell tissue according to claim 1, further comprising the step of dissolving the retaining material constituting each of the plurality of blocks housed in the divided housing to recover the plurality of cell tissues.

3. A method for producing cell tissue according to claim 1 or 2, further comprising the step of separating the plurality of blocks.

4. The method for producing cell tissue according to claim 3, wherein the step of separating the plurality of blocks includes the step of storing each of the plurality of blocks in a recovery housing having a plurality of chambers having a width wider than the spacing between the blades that divide the retaining material in the step of obtaining the plurality of blocks.

5. The method for producing cell tissue according to claim 3, wherein the step of separating the plurality of blocks includes the step of storing the plurality of blocks in a collection housing having a plurality of rooms such that each of the plurality of blocks is not stored in a room adjacent to one another.

6. A culture apparatus for bioprinting to obtain multiple cell tissues by culturing multiple cells separately, The culture apparatus described above is A culture enclosure is provided in the order of: a first chamber in which a first substrate for supporting the plurality of cells is arranged; a containment chamber configured to contain a culture medium; and a second chamber in which a second substrate for supporting the plurality of cells is arranged. A replacement means for replacing the solution contained in the aforementioned containment chamber with a soluble liquid retaining material that hardens under predetermined conditions, Equipped with a means for separation, The aforementioned plurality of cells are printed linearly from the first chamber to the second chamber, The separation means separates each of the plurality of cell tissues contained in the containment chamber from at least one of the substrates, the first substrate located in the first chamber and the second substrate located in the second chamber. The culture apparatus further comprises a dividing means for dividing the holding material along the direction of extension of the plurality of cell tissues held by the hardened holding material to obtain a plurality of blocks.

7. The culture apparatus according to claim 6, further comprising a dissolution means for dissolving the retaining material and recovering the plurality of cell tissues.

8. The culture apparatus according to claim 6 or 7, further comprising a means for separating the plurality of blocks.

Images