Method for producing cell tissue and culture apparatus

Abstract

To provide a method for producing a cell tissue, capable of preventing the cell tissue from being damaged, and having good recovery efficiency, and to provide a culture apparatus.SOLUTION: The method for producing a cellular tissue includes a step of filling a space between a first substrate and a second substrate with a soluble liquid holding material that cures under predetermined conditions, a step of curing the filled holding material, and a step of cutting out the cured holding material from the first substrate and the second substrate to separate each of at least one cellular tissue from at least one substrate of the first substrate and the second substrate.SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] The present disclosure relates to a method for producing cellular tissue and a culture device. [Background technology]

[0002] Research is being conducted into the technology of bioprinting tissues, with the aim of using the resulting tissues as edible steaks or processed meats.

[0003] Non-Patent Document 1 develops tendon gel integrated bioprinting and discloses a technique for assembling fibrous cell tissue created by tendon gel integrated bioprinting to construct artificial steak-like tissue in vitro. The tendon gel integrated bioprinting disclosed in Non-Patent Document 1 is a technique in which cells are printed onto a gel that mimics tendon, and then cultured with both ends of the cells attached to a tendon-mimicking support.

[0004] To achieve such a culture technique, Patent Document 1 discloses a method in which cells are arranged linearly with both ends attached to a support, and muscle cells are cultured in this state.

[0005] Furthermore, Patent Document 2 discloses a method in which a bioink containing cells is printed linearly into a supporting bath, the gel in the supporting bath is dissolved after printing, the solution in the supporting bath is removed, and a culture medium is added to culture the cells. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] International Publication No. 2021 / 193980 [Patent Document 2] International Publication No. 2021 / 193981 [Non-patent literature]

[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) Summary of the Invention [Problem to be solved by the invention]

[0008] By culturing cells with both ends supported on a substrate, the cells can be cultured under a condition in which tensile tension is generated between the substrate and the cells. The generation of tensile tension facilitates the formation of muscle fibers, muscle tissue, or sarcomere structures, and the resulting cell tissue more closely resembles animal muscle.

[0009] Although it is preferable to culture cells with both ends supported on a substrate, the substrate must be removed to recover the resulting cell tissue after culture. However, cell tissue is very fragile, and therefore, the cell tissue can be damaged when the substrate is removed, resulting in a problem of reduced recovery efficiency.

[0010] An object of the present disclosure is to provide a method and a culture device for producing cell tissue that prevents damage to the cell tissue and allows for efficient collection. [Means for solving the problem]

[0011] The method for producing at least one cellular tissue of the present disclosure includes the steps of linearly printing at least one cell between a first substrate and a second substrate such that both ends of the cell are supported by the first substrate and the second substrate, culturing the printed at least one cell to obtain at least one cellular tissue, filling the space between the first substrate and the second substrate with a soluble liquid retention material that hardens under predetermined conditions, hardening the filled retention material, and cutting the hardened retention material from at least one of the first substrate and the second substrate to separate each of the at least one cellular tissue from at least one of the first substrate and the second substrate.

[0012] The culture device disclosed herein is a culture device for bioprinting for culturing at least one cell to obtain at least one cellular tissue. The culture device includes a housing having, in this order: a first chamber in which a first substrate for supporting at least one cell is disposed; a storage chamber configured to store a culture solution; and a second chamber in which a second substrate for supporting at least one cell is disposed; a replacement means for replacing the solution contained in the storage chamber with a soluble liquid retention material that hardens under predetermined conditions; and a detachment means. The at least one cell is printed linearly from the first chamber toward the second chamber. The detachment means detaches each of the at least one cellular tissue contained in the storage chamber from at least one of the first substrate disposed in the first chamber and the second substrate disposed in the second chamber. [Effects of the Invention]

[0013] According to the present disclosure, the hardened retention material allows the cellular tissues to be separated from the substrate while retaining each piece of cellular tissue, thereby preventing damage to the cellular tissues and improving the efficiency of cellular tissue recovery. [Brief explanation of the drawings]

[0014] [Figure 1] FIG. 1 is a schematic diagram of a production method showing up to the culture step. [Figure 2] FIG. 1 is a schematic diagram of the production method showing the steps subsequent to the culturing step. [Figure 3] FIG. 1 is a diagram illustrating a schematic structure of a culture device according to one embodiment. [Figure 4] FIG. 10 is a plan view of the wall surface from the normal direction of the flat plate surface. [Figure 5] 1A and 1B are diagrams illustrating a printing process and a separation process using a culture device. [Figure 6] FIG. 10 is a plan view of a flat surface of a wall according to a modified example. [Figure 7] FIG. 10 is a diagram schematically illustrating the structure of a culture device according to a modified example. [Figure 8] 10A and 10B are schematic diagrams illustrating a manufacturing method according to a modified example. DETAILED DESCRIPTION OF THE INVENTION

[0015] Hereinafter, embodiments of the present disclosure will be described in detail with reference to the drawings. In the drawings, the same or corresponding parts are designated by the same reference numerals, and description thereof will not be repeated.

[0016] [Method of manufacturing 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 showing up to the culture step. Figure 2 is a schematic diagram of the production method showing steps after the culture step.

[0017] The cell tissue is obtained by culturing linearly printed cells. The cells are not particularly limited as long as they form fibrous cell tissue when printed linearly and cultured. The cells may be, for example, cells derived from an animal, and may be cells derived from a human or a non-human animal. The cells used in this embodiment are, for example, skeletal muscle cells. The skeletal muscle cells may be cells derived from muscle or cells derived from stem cells.

[0018] Referring to Figures 1 and 2, the method for producing cell tissue in 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 hardening step S7, a separation step S8, and a dissolution step S9.

[0019] (Base material / support material placement process S1) The substrate 1, the support material 3, and the substrate 2 are arranged in this order in the culture device. That is, the support material 3 is arranged so as to be sandwiched between the substrate 1 and the substrate 2.

[0020] The substrates 1 and 2 may be made of any material appropriately selected by those skilled in the art, as long as they are capable of binding to and supporting the cells printed in the printing step S2. The substrates 1 and 2 may be in either a solid or liquid form. The term "solid" includes a gel form. The physical properties of the substrates 1 and 2 are not particularly limited. For example, the substrates 1 and 2 may have physical properties that change shape under predetermined conditions, or may have physical properties that do not change shape. The predetermined conditions include, for example, temperature, pressure, electrical stimulation, pH, etc. The substrates 1 and 2 may be, for example, collagen or collagen nanofiber. The type of collagen is not particularly limited. For example, the substrates 1 and 2 may be made of multiple types of collagen of different types. Furthermore, the substrates 1 and 2 may have different compositions. In this embodiment, the substrates 1 and 2 are described as being collagen nanofibers.

[0021] The support material 3 is made of a soluble material. For example, the support material 3 is a solution of a polymeric substance such as gelatin, agar, or gellan gum dissolved in an aqueous solvent. In this case, in the substrate / support material placement step S1, the support material 3 may be in a gel or sol form. The support material 3 may have thixotropy, meaning that its viscosity decreases and becomes liquid when force is applied and gradually recovers when the force is removed. The support material 3 may also be a mixed solution prepared by, for example, crushing a gelled sample and dispersing it in a solvent such as a liquid medium. The gelled sample is prepared by dissolving a polymeric substance such as gelatin, agar, or gellan gum in an aqueous solvent and gelling it. In the substrate / support material placement step S1, the support material 3 is described as a mixed solution prepared by crushing a gelled sample from a gelatin solution and dispersing it in a liquid medium.

[0022] In this embodiment, as an example, after disposing a liquid substrate 1, the substrate 1 is partially cured to increase its viscosity, and then a support material 3 made of a mixed solution in which a gelled sample is dispersed in a solvent is disposed on the substrate 1. Then, the liquid substrate 2 is disposed on the support material 3. The method for disposing each sample is not particularly limited.

[0023] (Printing process S2) The printing step S2 is a step of printing cells C linearly. At this time, cells C are printed so that one end of cells 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. Bioink containing cells C is filled into a syringe 102 having a nozzle 103 attached to its tip. Nozzle 103 is inserted from substrate 2 toward substrate 1 so that the tip of nozzle 103 is positioned within substrate 1. By ejecting bioink from nozzle 103 while moving the tip of nozzle 103 in the direction from substrate 1 toward substrate 2, cells C are printed linearly from substrate 1 toward substrate 2.

[0024] Cells are printed in the support material 3. If the support material 3 is thixotropic, the viscosity decreases during printing due to stress generated by the movement of the nozzle 103 and the ejection of the bioink, but recovers after printing. Because the viscosity decreases during printing, the nozzle can be easily moved and the bioink can be easily ejected. However, the viscosity recovers and increases after printing, so the shape of the printed cells C is maintained by the support material 3 and the cells C are protected. Note that if the support material 3 is a solution in which a polymer substance is dissolved in an aqueous solvent, the support material 3 may be printed in a sol state, and then converted to a gel state after printing.

[0025] The printing step S2 may be performed automatically or semi-automatically using a known three-dimensional bioprinting device. Alternatively, a multi-nozzle dispenser having multiple nozzles may be used to print multiple cells at once.

[0026] The number of linear cells C printed in the printing process S2 is not particularly limited as long as it is one or more, and can be two or more, ten or more, 100 or more, 1,000 or more, or 10,000 or more, and is selected appropriately depending on the final product desired to be obtained and the size of the culture device, etc.

[0027] The diameter of the line formed by the cells C when printed is not particularly limited and is appropriately selected depending on the type of cells C, the viscosity of the bioink, the desired cell tissue, 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, it is sufficient that the multiple cells C are all printed linearly from the substrate 1 to the substrate 2, and the extension directions of the multiple cells C may or may not be parallel to each other. Note that in this embodiment, each of the multiple cells C is printed so that the extension directions of the multiple cells C are parallel to each other.

[0029] (Support process S3) The support step S3 is a step of hardening the substrates 1 and 2 to form substrates 1A and 2A, and supporting both ends of the cell C on the substrates 1A and 2A. In Figures 1 and 2, to clearly show that the shape of the substrates 1 and 2 has changed from a liquid state to a solid state, the solid substrates are represented by the symbols 1A and 2A and 1A and 2A, respectively, and the liquid substrates are represented by the symbols 1A and 2. The term "solid" includes a gel state, and hardening the substrates 1 and 2 refers to changing the shape of the substrates 1 and 2 into a shape suitable for supporting the ends of the cell C.

[0030] The substrates 1A and 2A may have a reversible property of changing from a solid state to a liquid state, or may have an irreversible property of not changing from a solid state to a liquid state.

[0031] When the shapes of the substrates 1 and 2 gradually change from a liquid state to a solid state, the printing step S2 and the supporting step 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 step S2 and the supporting step S3, at least one cell C is printed linearly so that both ends of the cell C are supported by the substrates 1 and 2, respectively.

[0032] Alternatively, the solid (gel) in the support material 3 may be converted to a liquid (sol) while the substrates 1 and 2 are hardened. For example, if the substrates 1 and 2 do not dissolve under conditions under which the solid (gel) in the support material 3 is converted to a liquid (sol), the culture device may be controlled to achieve these conditions and conditions for hardening the substrates 1 and 2, thereby converting the solid (gel) in the support material 3 to a liquid (sol) while hardening the substrates 1 and 2. By converting the solid (gel) in the support material 3 to a liquid (sol), the support material 3 can be easily replaced with a culture medium in the subsequent first replacement step S4. In FIG. 1, the support material containing the solid (gel) is represented by the symbol 3, and the support material after the transition to a liquid (sol) is represented by the symbol 3A.

[0033] In this embodiment, the substrates 1 and 2 are collagen nanofibers, and the support material 3 is a mixed solution prepared by pulverizing a gelled sample of gelatin solution and dispersing it in a liquid medium. By maintaining the temperature inside the culture device below a temperature at which collagen does not denature but above a temperature at which the gelatin in the support material 3 turns into a sol, the substrates 1 and 2 can be hardened while the gel in the support material 3 can be transformed into a sol. For example, the temperature inside the culture device can be maintained at a temperature below about 40°C, above 25°C, above 35°C, or above 37°C. Maintaining the inside of the culture device within this temperature range causes the substrates 1 and 2 to harden while the gelatin in the support material 3 dissolves. It is preferable that the temperature inside the culture device be maintained at 30°C to 40°C, more specifically, at 37°C, in consideration of the effect on the growth of the cell C.

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

[0035] The culture medium 4 can be appropriately selected by those skilled in the art depending on the cell type, etc. For example, the culture medium 4 may contain a differentiation-inducing factor for inducing differentiation of the cells.

[0036] Although the supporting step S3 and the step of dissolving the supporting material 3A are performed in parallel, they may be performed independently. For example, the step of dissolving the supporting material 3A may be performed after the supporting step S3, and then the first replacing step S4 may be performed.

[0037] (Culture step S5) The culturing step S5 is a step of culturing cells C in a culture medium 4 to obtain a cellular tissue T. The culturing conditions are appropriately designed by a person skilled in the art depending on the type of cell and the type of cellular tissue to be obtained. Furthermore, a first replacement step S4 may be performed during the culturing step S5 to replace the culture medium.

[0038] (Second substitution step S6) 2, the second replacement step S6 is a step of replacing the culture solution 4 with the retention material 5, filling the space between the substrate 1 and the substrate 2 with the retention material 5, which is a soluble liquid that hardens under predetermined conditions. For example, the retention material 5 is inserted between the substrates 1A and 2A from a syringe 106, and the culture solution 4 is discharged from a waste liquid port 107.

[0039] The retention material 5 is made of a soluble material and is handled in a dissolved liquid state in the second substitution step S6. The retention material 5 is a polymeric substance such as gelatin, agar, or gellan gum dissolved in an aqueous solvent. In the present embodiment, the retention material 5 is gelatin dissolved in an aqueous solvent. The retention material 5 preferably has a hardness that allows the retention material 5 to retain the cell tissue T when hardened. For example, when gelatin with a jelly strength of 300 g is used, the retention material 5 is a gelatin solution in which the gelatin content is adjusted to a range of 35 to 45 mg / mL, more specifically, a gelatin solution in which the gelatin content is adjusted to 40 mg / mL.

[0040] The support material 3 and the holding material 5 have in common the fact that they are both samples containing a polymeric substance. In this embodiment, the support material 3 is a solution in which a sample of a gelled polymeric substance is dispersed in a solvent, whereas the holding material 5 is a solution in which the polymeric substance is completely dissolved and turned into a sol.

[0041] The retention material 5 may be used as the culture solution used in the culture step S5. For example, a polymeric substance such as gelatin may be dissolved in the culture solution 4, and the cells C may be cultured using this solution. In this case, it is not necessary to perform the second replacement step S6. That is, a step of filling the retention material 5 may be performed before or in parallel with the culture step S5. Furthermore, in the second replacement step S6, the culture solution 4 is replaced with the retention material 5, but the retention material 5 may be replaced with a solution other than the culture solution 4. That is, a pretreatment step for increasing the strength of the cellular tissue T, or a step of washing the cellular tissue T, may be performed before the second replacement step S6.

[0042] (Curing process S7) The hardening step S7 is a step of hardening the retention material 5. In FIG. 2, to clearly show that the shape of the retention material 5 has changed from liquid to solid, the liquid retention material is represented by a symbol 5 and the solid retention material is represented by a symbol 5A. For example, the temperature inside the culture device is kept below 20°C, below 10°C, or below 5°C. The hardening conditions vary depending on the type of retention material 5, and are therefore designed according to that type.

[0043] (Separation process S8) The separation step S8 is a step of separating the base materials 1A, 2A from the cellular tissue T by separating the hardened retention material 5A from the hardened base materials 1A, 2A. At this time, the cellular tissue T is separated from the base materials 1A, 2A while being protected by the retention material 5A. Therefore, tensile force is less likely to be applied to the cellular tissue T when separating, and damage to the cellular tissue T can be prevented. This allows the recovery rate of the cellular tissue T to be increased.

[0044] In this embodiment, the separation step S8 may be performed by removing the entire block consisting of the substrates 1A, 2A and the retention material 5A from the culture device 100. Even during removal, the cellular tissue T is protected by the retention material 5A, so that damage to the cellular tissue T can be prevented and the recovery rate of the cellular tissue T can be increased.

[0045] (Dissolution step S9) The dissolution step S9 is a step of dissolving the retention material 5A. This allows the cell tissue T to be recovered. The dissolution conditions vary depending on the type of retention material 5A, and are therefore designed according to the type. For example, if the retention material 5A dissolves due to temperature, the retention material 5A containing the cell tissue T can be dissolved by pouring water of a predetermined temperature onto the retention material 5A, and the cell tissue T can be recovered. Also, if the retention material 5A dissolves due to pH, the retention material 5A can be dissolved by pouring a solution of a predetermined pH (e.g., a buffer solution) onto the retention material 5A, and the cell tissue T can be recovered.

[0046] More specifically, when gelatin is used as the material for retention material 5A, water at a temperature of less than about 40°C, 25°C or higher, 35°C or higher, or 37°C or higher is applied to retention material 5A. When gellan gum is used as the material for retention material 5A, a cationic solution such as Tris-HCl buffer, Tris-maleic acid buffer, or Bis-Tris buffer is applied to retention material 5A. When agar is used as the material for retention material 5A, water at a temperature of 80°C or higher is applied to retention material 5A.

[0047] In consideration of the effect on the cellular tissue T, the retention material 5A is preferably composed of a material that dissolves at temperatures below 40°C or a material that dissolves at a pH of 6.8 to 7.2. However, if the dissolved retention material 5A can be separated from the cellular tissue T simultaneously with the dissolution of the retention material 5, the effect on the cellular tissue T can be reduced. Therefore, the retention material 5 is not limited to one that dissolves under the above conditions, and may be one that dissolves under conditions that affect the cellular tissue T.

[0048] Furthermore, in this embodiment, a plurality of cells C are printed in the printing step S2 to obtain a plurality of cellular tissues T. The plurality of cells C are each printed linearly from the substrate 1 toward the substrate 2. Therefore, the plurality of cellular tissues T are held in the holding material 5A with their extension directions aligned. Therefore, as shown in FIG. 2, when dissolving the holding material 5A, it is preferable to place the holding material 5A with the surface on which the holding material 5A is placed aligned with the extension direction of the cellular tissues T, and then dissolve the holding material 5A. This allows the plurality of cellular tissues T to be recovered with their extension directions aligned. As a result, the process of assembling the recovered cellular tissues T does not require a process of aligning the fiber direction of the cellular tissues T.

[0049] In this embodiment, in the separation step S8, both the substrates 1A and 2A are separated from the cellular tissue T. Note that in the separation step S8, at least one of the substrates 1A and 2A may be separated. When a material that does not dissolve in the dissolving step S9 is used as the substrate, one end of the cellular tissue T is retained even after the dissolving step S9, which may make it easier to handle multiple cellular tissues T together.

[0050] [Culture equipment for realizing the separation process] 3 is a diagram showing a schematic structure of a culture device according to one embodiment. The culture device 100 is a culture device for bioprinting to culture cells and obtain cellular tissue. The culture device 100 includes a housing 10, a replacement means 20, and a detachment means 160.

[0051] The housing 10 is provided with, in that order, a first chamber 110 in which the substrate 1 is placed, a storage chamber 120 configured to be able to store a culture solution 4, and a second chamber 130 in which the substrate 2 is placed.

[0052] The culture device 100 is configured so that a substrate can be placed in the first chamber 110 and the second chamber 130. The culture device 100 may be provided with a substrate already placed in each of the first chamber 110 and the second chamber 130, or may be configured so that a substrate can be inserted and placed in each of the first chamber 110 and the second chamber 130 later.

[0053] The replacement means 20 replaces the sample (solution) filling the storage chamber 120 with another sample (solution). Specifically, the replacement means 20 includes an insertion port 22 configured to allow attachment of a tube with a tube pump attached, a syringe, or the like, and an outlet 24 for discharging the sample in the storage chamber 120 to the outside of the culture device 100. The insertion port 22 and the outlet 24 are each connected to the storage chamber 120.

[0054] For example, in a first substitution step S4, the substitution means 20 substitutes the sample filling the storage chamber 120 from the support material 3A with the culture solution 4. In addition, in a second substitution step S6 after the cells C are cultured in the culture step S5 to obtain the cellular tissue T, the substitution means 20 substitutes the sample filling the storage chamber 120 from the culture solution 4 with the retention material 5.

[0055] The detaching means 160 is a device for detaching the cellular tissue T obtained by culturing in the storage chamber 120 from the substrate. The detaching means 160 includes a first wall surface 161 and a second wall surface 162. The first wall surface 161 is a wall that separates the first chamber 110 and the storage chamber 120, and is disposed within the housing 10. The second wall surface 162 is a wall that separates the storage chamber 120 and the second chamber 130, and is disposed within the housing 10. The first wall surface 161 is disposed within the housing 10 so that the flat surface of the first wall surface 161 faces the first chamber 110. The second wall surface 162 is disposed within the housing 10 so that the flat surface of the second wall surface 162 faces the second chamber 130.

[0056] Both first wall surface 161 and second wall surface 162 are configured to be slidable. More specifically, first wall surface 161 is configured to be slidable along the flat surface of first wall surface 161. Second wall surface 162 is configured to be slidable along the flat surface of second wall surface 162. In FIG. 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-axis and Z-axis is the Y-axis. In this embodiment, first wall surface 161 and second wall surface 162 are arranged parallel to each other within housing 10 and have the same sliding direction. The sliding direction of first wall surface 161 and the sliding direction of second wall surface 162 may be different.

[0057] In a state where a substrate is disposed in each of the first chamber 110 and the second chamber 130, the first wall surface 161 can be said to be disposed in the culture device 100 so that the flat surface faces the substrate. Similarly, the second wall surface 162 can be said to be disposed in the culture device 100 so that the flat surface faces the substrate.

[0058] Fig. 4 is a plan view of the wall surface viewed from the normal direction of the flat surface. Note that, hereinafter, 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 the wall surface 16. The plan view shown in Fig. 4 is a view obtained by viewing the flat surface of the wall surface 16 from the normal direction (Z-axis direction) of the flat surface of the wall surface 16.

[0059] 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 10,000 or more. By forming the openings 60 in the first wall surface 161 and the second wall surface 162, the printing step S2 can be carried out without removing the first wall surface 161 and the second wall surface 162.

[0060] The first wall surface 161 and the second wall surface 162 may be plate-like members without openings 60. In this case, after the substrates 1, 2 and the support material 3 are placed in the culture device 100, the first wall surface 161 and the second wall surface 162 are slid to eliminate the gap between the storage chamber 120 and the first chamber 110 and the second chamber 130, and the printing process is performed. Thereafter, in the separation process, the first wall surface 161 and the second wall surface 162 may be slid between the storage chamber 120 and the first chamber 110 and the second chamber 130, and the cell tissue T may be separated from the substrates 1, 2.

[0061] The size of opening 60 need only be large enough to allow a nozzle for ejecting bioink containing cells to pass through, and if opening 60 is circular, the diameter of opening 60 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.

[0062] It is preferable that the first wall surface 161 and the second wall surface 162 are arranged in the culture device 100 so 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.

[0063] 4, a plurality of openings 60 may be formed in a two-dimensional array on the wall surface 16. The cells C are printed so that at least one cell C passes through each of the two-dimensionally arranged plurality of openings 60. In this way, by forming the plurality of openings 60 in a two-dimensional array on the wall surface 16, each cell tissue T can be easily handled in the procedure after the cellular tissue T is separated from the substrates 1 and 2.

[0064] The plurality of openings 60 may be arranged in two directions that intersect with each other. The crossing angle is 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 Fig. 4, the plurality of openings 60 are arranged in two directions that intersect with each other at right angles. The plurality of openings 60 may also be arranged in a staggered pattern.

[0065] FIG. 5 illustrates the printing and separation processes using a culture device. In the printing process S2A, the nozzle 103 for ejecting bioink containing the cells C is moved as follows, allowing the cells C to 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. First, the nozzle 103 is positioned so that it passes through the opening 60 in the second wall surface 162 and the opening 60 in the first wall surface 161. Next, while ejecting the bioink from the nozzle 103, the nozzle 103 is moved so that the tip of the nozzle 103 passes from the opening 60 in the first wall surface 161 to the opening 60 in the second wall surface 162. This allows the cells C to be arranged linearly so that they pass from the opening 60 in the first wall surface 161 to the opening 60 in the second wall surface 162. As a result, the cells C are arranged linearly from the substrate 1 arranged in the first chamber 110 to the substrate 2 arranged in the second chamber 130. Thereafter, the base materials 1 and 2 are hardened, whereby both ends of the linear cell C can be supported by the base materials 1 and 2.

[0066] Next, in the separation step S8A, the first wall surface 161 and the second wall surface 162 are each slid in the X-axis direction along the flat plate surface. As a result, the cellular tissue T arranged to pass through the opening 60 is cut by the edge of the opening 60. As a result, the cellular tissue T is separated from the substrates 1A and 2A.

[0067] In this way, the cell tissue T can be separated from the substrates 1 and 2 simply by sliding the first wall surface 161 and the second wall surface 162, respectively, and therefore the separation operation can be easily performed.

[0068] The arrangement pattern and shape of openings 60 are not limited to those shown in Fig. 4. Fig. 6 is a plan view of the flat surface of a wall surface according to a modified example. Wall surface 16A is formed with openings 60A that are elongated and whose length along the X-axis direction (sliding direction) is longer than its length along the Y-axis direction perpendicular to the X-axis direction (sliding direction).

[0069] When the opening 60A has an elongated shape, it is easier to align multiple nozzles, such as a multi-nozzle dispenser, when inserting them into the culture device 100 at once, compared to when multiple openings 60 are formed in rows and columns. Furthermore, because the length along the X-axis direction is shorter than the length along the Y-axis direction, the distance from the cellular tissue T to the edge of the opening 60A can be shortened. This shortens the distance over which the wall surface 16A slides when separating the substrate from the cellular tissue T.

[0070] Furthermore, although the storage chamber 120 is divided into the first chamber 110 and the second chamber 130 by the first wall surface 161 and the second wall surface 162, the storage chamber 120 may be formed by placing a box inside the housing 10. FIG. 7 is a diagram schematically showing the structure of a culture device according to a modified example. The culture device 100B may include a box 62 placed within the housing 10 so as to be sandwiched between predetermined spaces (the first chamber 110 and the second chamber 130). The interior of the box 62 corresponds to the storage chamber 120. An opening is formed in each of the first wall surface 161B, which forms the boundary between the box 62 and the first chamber 110, and the second wall surface 162B, which forms the boundary between the box 62 and the second chamber 130. The shape of the opening is not particularly limited, and openings having the shapes shown in FIG. 4 or FIG. 6 are formed in the first wall surface 161B and the second wall surface 162B.

[0071] The first wall surface 161B and the second wall surface 162B are each configured to be slidable along the flat surface of the wall surface, and at least one of the first wall surface 161B and the second wall surface 162B is configured to be open in the Z-axis direction perpendicular to the wall surface.

[0072] By configuring at least one of first wall surface 161B and second wall surface 162B to be openable and closable like a lid, the retention material containing the cellular tissue can be easily removed from culture device 100B after the cellular tissue has been separated from the substrate, making it easy to handle each cellular tissue in the procedure after the cellular tissue has been separated from the substrate.

[0073] Although the first wall surface 161 and the second wall surface 162 separating the chambers in the housing 10 are defined as the separation means 160, 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 housing 10 so that a flat plate can be inserted into the storage chamber 120 in a direction (X-axis direction or Y-axis direction) perpendicular to the extension direction (Z-axis direction) of the cellular tissue T. In this case, the first wall surface 161 and the second wall surface 162 may not be provided.

[0074] [Variations] In the above embodiment, after the substrates 1A and 2A are separated from the cellular tissue T, the lump of retention material 5A is dissolved in the dissolution step S9. Note that before dissolving the retention material 5A, the retention material 5A may be divided into multiple blocks and then the blocks may be dissolved. Figure 8 is a schematic diagram of a manufacturing method according to a modified example. Note that the manufacturing method according to the modified example shares the steps up to the separation step with the manufacturing method shown in Figures 1 and 2, so Figure 8 only shows the steps from the separation step onwards.

[0075] The manufacturing method according to the modified example differs from the manufacturing method shown in FIGS. 1 and 2 in that it further includes a dividing step S92 and an aligning step S94, and in that it includes a dissolving step S9A instead of the dissolving step S9.

[0076] (Dividing process S92) In the dividing step S92, the hardened retention material 5A is divided along the extension direction of the cellular tissue T to obtain a plurality of blocks 50. At least one of the plurality of blocks 50 contains at least one cellular tissue T. The number of cellular tissues T to be included in each block 50 can be appropriately selected by those skilled in the art and is determined, for example, depending on the subsequent processing. The number of blocks 50 may be two or more. For example, when cells C are printed in two mutually orthogonal directions as shown in FIG. 4, the retention material 5A may be divided into one row or two or more rows.

[0077] By dividing the retention material 5A, the cellular tissue can be handled in units of blocks 50, and the cellular tissue is easier to handle because it is protected by the hardened retention material 5A that makes up the block 50. Also, by dissolving the block 50, it is easy to obtain a bundle consisting of one or more cellular tissues T. Furthermore, when combining multiple types of cellular tissues T to create an aggregate that mimics the structure of a specific tissue, it is easier to perform the combination work by combining the block 50 units than by combining the cellular tissues T individually.

[0078] (Alignment step 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 extension directions of the cellular tissues T are aligned with one another. At this time, each of the plurality of blocks 50 may be arranged on a mesh 200 having a wave-like structure. When arranging the blocks 50 on the mesh 200 having a wave-like structure, the blocks 50 may be arranged in the recesses 220 so that the extension direction of the recesses 220 and the extension direction of the cellular tissues T contained in the blocks 50 are aligned.

[0079] (Dissolution process S9A) The dissolving step S9A is performed after the alignment step S94. After arranging the multiple blocks 50 so that the extension directions of the cellular tissues T are aligned, the holding material 5A is dissolved, thereby obtaining multiple cellular tissues T with aligned extension directions. Therefore, there is no need to align the extension directions of the cellular tissues T with tweezers or the like, and the risk of damaging the cellular tissues T can be reduced. Note that the dissolving conditions are the same as those in the above embodiment, so explanations will be omitted.

[0080] Furthermore, when the block 50 is placed on the mesh 200 having a wavy structure and then dissolved, the cellular tissue T is collected in the recesses 220, and bundles of the cellular tissue T can be easily obtained.

[0081] [Aspect] It will be understood by those skilled in the art that the above-described embodiments are specific examples of the following aspects.

[0082] (Item 1) According to one embodiment, a method for producing at least one cellular tissue includes the steps of linearly printing at least one cell between a first substrate and a second substrate such that both ends of the cell are supported by the first substrate and the second substrate, culturing the printed at least one cell to obtain at least one cellular tissue, 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, and cutting the hardened retaining material from at least one of the first substrate and the second substrate to separate each of the at least one cellular tissue from at least one of the first substrate and the second substrate.

[0083] According to the method for producing cellular tissue described in paragraph 1, the cellular tissues are separated from the first substrate and the second substrate while each of the cellular tissues is held in place by the hardened holding material, thereby preventing damage to the cellular tissues and improving the efficiency of recovering the cellular tissues.

[0084] (Item 2) In the method for producing cellular tissue described in Item 1, a flat-plate-shaped first member and a flat-plate-shaped second member can be arranged in this order between a first substrate and a second substrate. The flat surface of the first member and the flat surface of the second member are arranged facing the first substrate and the second substrate, respectively. The first member is configured to be slidable along the flat surface of the first member. The second member is configured to be slidable along the flat surface of the second member. In the separating step, the first member and the second member are slid to separate at least one cellular tissue from the first substrate and the second substrate, respectively.

[0085] According to the method for producing cell tissue described in item 2, the cell tissue can be separated from the substrate simply by sliding the first member and the second member, respectively, and therefore the separation operation can be easily performed.

[0086] (Item 3) In the manufacturing method described in Item 2, the first member has at least one first opening for passing at least one cell. The second member has at least one second opening for passing at least one cell. In the printing step, each of the at least one cell is printed so as to pass through the at least one first opening and the at least one second opening.

[0087] According to the method for producing cell tissue described in item 3, openings are formed in the first member and the second member, so that cells can be printed without removing the first member and the second member.

[0088] (Item 4) In the method for producing cellular tissue described in any one of Items 1 to 3, in the printing step, a plurality of cells are each printed in a linear shape. In the step of obtaining at least one cellular tissue, the plurality of cells are each cultured to obtain a plurality of cellular tissues. The production method further includes a step of dividing the hardened retention material along the extension direction of the cellular tissues retained in the retention material to obtain a plurality of blocks.

[0089] According to the manufacturing method described in paragraph 4, the cellular tissue can be handled in blocks, and is easy to handle because the cellular tissue is protected by the hardened retaining material that makes up the blocks. Also, when combining multiple types of cellular tissue to create an aggregate that mimics the structure of a specific tissue, combining blocks makes it easier to perform the combining work than combining individual cellular tissues.

[0090] (Item 5) The method for producing cell tissue according to any one of items 1 to 4 further comprises the step of dissolving the cut-out retention material.

[0091] According to the manufacturing method described in item 5, the cell tissue can be collected by the simple method of dissolving the cut-out retention material.

[0092] (Item 6) The method for producing cellular tissue described in Item 4 further includes the steps of placing a plurality of blocks so as to align the extension direction of each of the plurality of cellular tissues, and dissolving the retaining material that constitutes each of the placed blocks.

[0093] According to the manufacturing method described in item 6, a plurality of cellular tissues can be obtained with the same extension direction by dissolving a block in which the cellular tissues are arranged in the same extension direction. This eliminates the need to align the extension direction of the cellular tissues with tweezers or the like, thereby reducing the risk of damaging the cellular tissues.

[0094] (Item 7) The method for producing a cellular tissue according to any one of items 1 to 6 includes, after obtaining at least one cellular tissue, carrying out a step of filling with a retention material.

[0095] (Item 8) One embodiment of the culture device is a culture device for bioprinting for culturing at least one cell to obtain at least one cellular tissue. The culture device includes a housing having, in that order, a first chamber in which a first substrate for supporting at least one cell is disposed, a storage chamber configured to store a culture solution, and a second chamber in which a second substrate for supporting at least one cell is disposed; a replacement means for replacing the solution contained in the storage chamber with a soluble liquid retention material that hardens under predetermined conditions; and a detachment means. The at least one cell is printed linearly from the first chamber toward the second chamber. The detachment means detaches each of the at least one cellular tissue contained in the storage chamber from at least one of the first substrate disposed in the first chamber and the second substrate disposed in the second chamber.

[0096] According to the culture device described in item 8, a retention material that hardens under predetermined conditions can be inserted into the storage chamber by the replacement means, and the cellular tissues can be separated from the substrate while each piece is held by the hardened retention material. This prevents damage to the cellular tissues and improves the efficiency of retrieving the cellular tissues.

[0097] (Item 9) In the culture device according to item 8, the separating means includes a first wall disposed within the housing and separating the first chamber from the storage chamber, and a second wall disposed within the housing and separating the second chamber from the storage chamber. The first wall is configured to be slidable along the flat surface of the first wall. The second wall is configured to be slidable along the flat surface of the second wall. The separating means separates the first substrate and the second substrate from each of the at least one cellular tissue by sliding the first wall and the second wall.

[0098] According to the culture device described in item 9, the cell tissue can be separated from the substrate simply by sliding the first wall surface and the second wall surface, respectively, and therefore the separation operation can be easily performed.

[0099] (Item 10) In the culture device according to item 9, the first wall surface has at least one first opening for allowing at least one cell to pass therethrough, and the second wall surface has at least one second opening for allowing at least one cell to pass therethrough.

[0100] According to the culture device described in item 10, cells can be printed linearly without removing the first wall surface and the second wall surface.

[0101] (Item 11) In the culture device according to item 10, the first wall has a plurality of first openings that are two-dimensionally arranged when the first wall is viewed in a plan view from the normal direction to the flat surface of the first wall. The second wall has a plurality of second openings that are two-dimensionally arranged when the second wall is viewed in a plan view from the normal direction to the flat surface of the second wall.

[0102] According to the culture device described in item 11, it is easy to arrange a plurality of cells in an aligned state, and each cell tissue can be easily handled in the procedure after the cell tissue is separated from the substrate.

[0103] (Item 12) In the culture device according to item 10, at least one first opening has an elongated shape in which, when the first wall is viewed in a plane from the normal direction to the flat surface of the first wall, the length along the sliding direction of the first wall is shorter than the length along the direction perpendicular to the sliding direction. At least one second opening has an elongated shape in which, when the second wall is viewed in a plane from the normal direction to the flat surface of the second wall, the length along the sliding direction of the second wall is shorter than the length along the direction perpendicular to the sliding direction.

[0104] According to the culture device described in paragraph 12, when multiple nozzles, such as a multi-nozzle dispenser, are inserted into the culture device at once, it is easier to align the position of the multi-nozzle dispenser compared to when multiple openings are formed in the matrix direction. Furthermore, because the length along the sliding direction is shorter, the distance from the cellular tissue to the edges of the first and second openings can be shortened. This shortens the distance over which the first and second wall surfaces must be slid when separating the first and second substrates from the cellular tissue.

[0105] The embodiments disclosed herein are intended to be combined as appropriate within the scope of any technical inconsistency. The embodiments disclosed herein should be considered to be illustrative and not restrictive in all respects. The scope of the present invention is defined by the claims, not by the description of the above-mentioned embodiments, and is intended to include all modifications within the meaning and scope of the claims. [Explanation of symbols]

[0106] 1,1A,2,2A substrate, 3,3A support material, 4 culture medium, 5,5A holding material, 10 housing, 16,16A wall surface, 20 replacement means, 22 insertion port, 24 discharge port, 50 block, 60,60A opening, 62 box, 100,100B culture device, 102,104,106 syringe, 103 nozzle, 105,107 waste liquid port, 110 first chamber, 120 storage chamber, 130 second chamber, 160 separation means, 161,161B first wall surface, 162,162B second wall surface, 200 mesh, 220 recess, C cell, T cell tissue.

Claims

1. 1. A method for producing at least one cellular tissue, comprising: printing at least one cell linearly between a first substrate and a second substrate such that both ends of the cell are supported by the first substrate and the second substrate, respectively; Culturing the printed at least one cell to obtain the at least one cell tissue; Filling a space between the first substrate and the second substrate with a soluble liquid retaining material that hardens under predetermined conditions; A step of hardening the filled retention material; and cutting the hardened retention material from at least one of the first substrate and the second substrate, thereby separating each of the at least one cellular tissue from at least one of the first substrate and the second substrate.

2. A flat plate-shaped first member and a flat plate-shaped second member can be arranged in this order between the first base material and the second base material, a flat surface of the first member and a flat surface of the second member are disposed opposite the first base material and the second base material, respectively; the first member is configured to be slidable along a flat surface of the first member, the second member is configured to be slidable along a flat surface of the second member, The method for producing a cellular tissue according to claim 1 , wherein in the separating step, each of the at least one cellular tissue is separated from the first substrate and the second substrate by sliding the first member and the second member.

3. the first member has at least one first opening formed therein for allowing the at least one cell to pass therethrough; the second member has at least one second opening formed therein for passage of the at least one cell; The method for producing a cellular tissue according to claim 2 , wherein in the printing step, each of the at least one cell is printed so as to pass through the at least one first opening and the at least one second opening.

4. In the printing step, a plurality of cells are printed in a linear shape, In the step of obtaining at least one cell tissue, the plurality of cells are cultured to obtain a plurality of cell tissues, The method for producing cellular tissue described in any one of claims 1 to 3, further comprising a step of dividing the hardened retention material along the extension direction of the cellular tissue retained in the retention material to obtain multiple blocks.

5. The method for producing cell tissue according to any one of claims 1 to 3, further comprising the step of dissolving the cut-out retention material.

6. placing the plurality of blocks so that the extension directions of the plurality of cellular tissues are aligned; The method for producing cell tissue according to claim 4 , further comprising the step of dissolving the retention material that constitutes each of the plurality of placed blocks.

7. The method for producing a cellular tissue according to any one of claims 1 to 3, further comprising the step of filling the retention material after obtaining the at least one cellular tissue.

8. A culture device for bioprinting for culturing at least one cell to obtain at least one cell tissue, comprising: a housing in which a first chamber in which a first substrate for supporting the at least one cell is disposed, a storage chamber configured to be able to store a culture solution, and a second chamber in which a second substrate for supporting the at least one cell is disposed are provided in this order; a replacement means for replacing the solution contained in the container with a soluble liquid-state retaining material that hardens under predetermined conditions; and a separating means; the at least one cell is printed linearly from the first chamber to the second chamber; The detachment means detaches each of the at least one cellular tissue contained in the storage chamber from at least one of the first substrate placed in the first chamber and the second substrate placed in the second chamber.

9. The separating means is a first wall surface disposed within the housing and separating the first chamber from the storage chamber; a second wall surface disposed within the housing and separating the second chamber from the storage chamber; The first wall surface is configured to be slidable along a flat surface of the first wall surface, The second wall surface is configured to be slidable along a flat surface of the second wall surface, The culture device according to claim 8 , wherein the detachment means detaches the first substrate and the second substrate from each of the at least one cellular tissue by sliding the first wall surface and the second wall surface.

10. the first wall surface has at least one first opening formed therein for allowing the at least one cell to pass therethrough; The culture device according to claim 9 , wherein the second wall surface has at least one second opening formed therein for allowing the at least one cell to pass therethrough.

11. a plurality of first openings that are two-dimensionally arranged when the first wall surface is viewed in a plan view from a normal direction of a flat plate surface of the first wall surface; The culture device according to claim 10, wherein the second wall surface has a plurality of second openings formed thereon that are two-dimensionally arranged when the second wall surface is viewed in a plan view from a normal direction to the flat surface of the second wall surface.

12. the at least one first opening has an elongated shape in which a length along a sliding direction of the first wall surface is shorter than a length along a direction perpendicular to the sliding direction when the first wall surface is viewed in a plan view from a normal direction of a flat plate surface of the first wall surface, The culture device according to claim 10, wherein, when the second wall surface is viewed in a plane from a normal direction to the flat surface of the second wall surface, the at least one second opening has an elongated shape in which the length along the sliding direction of the second wall surface is shorter than the length along a direction perpendicular to the sliding direction.

Images