Culturing apparatus and printing method

Abstract

To provide a technique capable of printing bioink to a desired length so as to obtain a linear cell tissue of a desired length.SOLUTION: A culturing apparatus includes a bioprinter that prints bioink in a linear form, a housing configured to be capable of accommodating a base material that supports the bioink, a driving device that relatively moves the bioprinter and the housing, and a control device. The control device, after positioning a discharge port of the bioprinter at a print start position in the housing, controls the driving device such that the discharge port moves in a first direction within the base material in the housing. The control device controls a bioink pump so as to start applying pressure to the bioink after the discharge port is inserted into the base material and before the discharge port starts movement in the first direction from the print start position.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present disclosure relates to a bioprinting culture device for linearly printing a cell-containing bioink to obtain linear cellular tissue, and a method for printing a cell-containing bioink that will become a cellular tissue upon culturing into a substrate that supports the bioink. [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] Patent Document 1 discloses printing a line of bioink in a printing bath composed of a supporting bath material and a collagen layer. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] International Publication No. 2021 / 193980 Summary of the Invention [Problem to be solved by the invention]

[0005] When printing the bioink linearly, a syringe is inserted into a substrate that supports cells three-dimensionally, and after the syringe nozzle reaches the printing start position, the bioink is ejected while the syringe is being pulled up. If the syringe pull-up operation is started simultaneously with the start of the bioink ejection operation, the substrate may get caught in the syringe when the syringe is inserted, and this may prevent the bioink printing from starting from the printing start position, resulting in a printed bioink line that is not the desired length, which could result in a failure to obtain cell tissue of the desired length.

[0006] An object of the present disclosure is to provide a technology that enables printing of bioink to a desired length in order to obtain linear cell tissue of a desired length. [Means for solving the problem]

[0007] The culture device disclosed herein is a culture device for bioprinting, which prints a line of bioink containing cells to obtain a line of cellular tissue. The culture device includes a bioprinter that ejects the bioink to print the bioink in a line, a housing configured to accommodate a substrate that supports the bioink, a drive device that moves the bioprinter and the housing relative to one another, and a control device. The bioprinter includes a tube that accommodates the bioink and a pump that applies pressure to the bioink in a direction toward the tube's outlet. The control device positions the outlet at a print start position within the housing that accommodates the substrate, and then controls the drive device to move the outlet in a first direction through the substrate within the housing. The control device controls the pump to begin applying pressure to the bioink in a direction toward the outlet after the outlet is inserted into the substrate and before the outlet begins moving in the first direction from the print start position.

[0008] The printing method disclosed herein is a method for printing a bioink containing cells that will become cellular tissue when cultured into a substrate that supports the bioink. The printing method includes the steps of positioning a discharge port of a bioprinter for printing the bioink linearly at a print start position within the substrate, moving the discharge port within the substrate from the print start position in a first direction, and, after the discharge port is inserted into the substrate but before the discharge port starts moving in the first direction, starting to apply pressure to the bioink within the bioprinter in a direction toward the discharge port. [Effects of the Invention]

[0009] According to the present disclosure, by starting to apply pressure to the bioink in a direction toward the ejection port before the ejection port starts moving in a first direction from the print start position, the bioink can be printed to a desired length. [Brief explanation of the drawings]

[0010] [Figure 1] FIG. 1 is a diagram illustrating a schematic structure of a culture device according to one embodiment. [Figure 2] FIG. 1 is a schematic cross-sectional view of a bioprinter. [Figure 3] FIG. 2 is a diagram illustrating an outline of a printing operation. [Figure 4] FIG. 10 is a schematic diagram showing an example of successful printing. [Figure 5] FIG. 10 is a schematic diagram showing an example of a failed print. [Figure 6] 4 is a flowchart showing a process executed by a control device. DETAILED DESCRIPTION OF THE INVENTION

[0011] 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.

[0012] [Culture equipment] FIG. 1 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, which prints bioink containing cells in a linear shape to obtain linear cellular tissue. The cells are not particularly limited as long as they can be printed in a linear shape and cultured to form fibrous cellular tissue. The cells may be, for example, cells derived from an animal, such as human cells or cells derived from an animal other than human. The cells used in this embodiment are, for example, skeletal muscle cells. The skeletal muscle cells may be muscle-derived cells or stem cell-derived cells.

[0013] The culture device 100 includes a housing 10, a bioprinter 20, a drive device 30, a control device 40, an input device 50, and a display device 60. The control device 40 controls the bioprinter 20 and the drive device 30. The control device 40 also receives input from the input device 50 and displays an image for receiving the input from the input device 50 on the display device 60.

[0014] The housing 10 is configured to be able to house a substrate that supports the bioink three-dimensionally. The substrate includes a first support material 1, a second support material 2, and a support material 3. The first support material 1, the support material 3, and the second support material 2 are housed in this order from the bottom side of the housing 10. In FIG. 1, the direction from the first support material 1 to the second support material 2 is the Z-axis direction, and the plane perpendicular to the Z-axis direction is the XY plane.

[0015] The culture device 100 may be provided with the substrate already placed inside the housing 10, or may be configured so that the substrate can be inserted and placed inside the housing 10 later. Furthermore, the inside of the housing 10 may be divided into areas by walls or the like where the substrates of the first support material 1, the second support material 2, and the support material 3 are placed. In this case, the walls separating the areas may have openings formed therein through which the tubes 22 of the bioprinter 20 can be inserted.

[0016] Both the first support material 1 and the second support material 2 support the end of the linear bioink when the bioink is printed linearly. Both the first support material 1 and the second support material 2 may be composed of a material appropriately selected by a person skilled in the art, as long as they bind to and support the cells contained in the bioink. The first support material 1 and the second support material 2 may be in either a solid or liquid form. A solid form includes a gel form. The physical properties of the first support material 1 and the second support material 2 are not particularly limited. For example, the first support material 1 and the second support material 2 may have physical properties that allow them to change shape under specified conditions, or may have physical properties that prevent them from changing shape. The specified conditions include, for example, temperature, pressure, electrical stimulation, and pH. The first support material 1 and the second support material 2 may be, for example, collagen or collagen nanofiber. The type of collagen is not particularly limited. For example, the first support material 1 and the second support material 2 may be composed of multiple types of collagen. Furthermore, the first support material 1 and the second support material 2 may have different compositions. In this embodiment, the first support material 1 and the second support material 2 will be described as being collagen nanofibers.

[0017] The support material 3 is made of a soluble material. For example, the support material 3 is a solution in which a polymeric substance such as gelatin, agar, or gellan gum is dissolved in an aqueous solvent. 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. A gelled sample is prepared by dissolving a polymeric substance such as gelatin, agar, or gellan gum in an aqueous solvent and gelling it. The support material 3 will be described as a mixed solution prepared by crushing a gelled sample from a gelatin solution and dispersing it in a liquid medium.

[0018] The bioprinter 20 includes a tube 22 that contains bioink, and a pump 24. The pump 24 is configured to apply pressure to the bioink contained in the tube 22 in a direction toward the discharge port 22a of the tube 22 (hereinafter also referred to as the "discharge direction D1"). The pump 24 is, for example, a plunger pump, and as described below, causes a piston to reciprocate, thereby sucking the sample (bioink) into the tube 22 or discharging the sample from the tube 22.

[0019] The drive unit 30 moves the housing 10 and the bioprinter 20 relative to one another. In this embodiment, the drive unit 30 is configured to move the bioprinter 20. The drive unit 30, for example, moves the bioprinter 20 horizontally and vertically. The drive unit 30 can freely move the bioprinter 20 using, for example, a solenoid actuator or a stepping motor.

[0020] The control device 40 is a device for controlling the entire culture device 100 and includes a processor 42 and a memory 44. The processor 42 is, for example, a central processing unit (CPU) and is an arithmetic circuit that executes predetermined arithmetic processing described in a program. The memory 44 includes non-volatile or volatile memory such as a read-only memory (ROM) or a random access memory (RAM), and / or a large-capacity storage device such as a hard disk drive (HDD) or a solid state drive (SSD). The processor 42 reads out the programs and data stored in the memory 44 and controls the pump 24, drive device 30, and other components that constitute the culture device 100.

[0021] [Bioprinter configuration] 2 is a schematic cross-sectional view of the bioprinter. The tube 22 is configured to be detachable from the pump 24, and includes a connection port 222 and a nozzle 224. The tube 22 can be attached to the pump 24 by attaching the connection port 222 to a connection port 242a of a pump housing 242 of the pump 24.

[0022] Pump 24 includes a pump housing 242 and a piston 244 configured to be able to reciprocate within pump housing 242. Pump housing 242 is configured such that, when piston 244 moves to a position closest to connection port 242a during reciprocating motion, a gap Ga of a predetermined volume is formed between piston 244 and open end 242b of connection port 242a.

[0023] The predetermined volume need only be equal to or greater than the volume of bioink contained in the tube 22 during one printing operation. In the example shown in Fig. 2, the pump housing 242 is formed with a container 242c that contains the piston 244 and a tip end 242d that communicates with the open end 242b. The inner diameter A1 of the container 242c is larger than the outer diameter A2 of the piston 244. The inner diameter A3 of the tip end 242d at the boundary with the container 242c is smaller than the outer diameter A2 of the piston 244. Therefore, when the piston 244 moves to a position closest to the connection port 242a during reciprocating motion, a void Ga equal to the volume of the tip end 242d is formed within the pump housing 242.

[0024] In this embodiment, the tube 22 is configured to be detachable from the pump 24, and therefore can be easily replaced. This makes it possible to make the tube 22 disposable and the pump 24 reusable, which is hygienic and economical. Furthermore, because a gap Ga is formed within the pump housing 242, it is possible to prevent bioink from seeping into the pump housing 242 when the bioink is sucked. This eliminates the need to clean and sterilize the inside of the pump 24 when the bioprinter 20 is used repeatedly.

[0025] In the following, the direction opposite to the discharge direction D1 is referred to as the suction direction D2. The pump 24 moves the piston 244 in the suction direction D2 with the discharge port 22a of the nozzle 224 placed in the bioink, thereby drawing the bioink into the nozzle 224. The pump 24 also moves the piston 244 in the discharge direction D1 with the bioink contained in the nozzle 224, thereby discharging the bioink from the discharge port 22a.

[0026] [Outline of printing operation] The sequence of operations involved in printing a bio-ink into a substrate will now be described with reference to Figure 3. Figure 3 is a diagram showing an outline of the printing operation.

[0027] With the bioink contained in the tube 22, the control device 40 controls the drive device 30 so that the discharge port 22a of the bioprinter 20 is positioned at the print start position Ps within the housing 10. Specifically, the control device 40 controls the pump 24 and the drive device 30 to suck a predetermined volume of bioink into the tube 22. Thereafter, the control device 40 controls the drive device 30 to move the tube 22 of the bioprinter 20 from the top of the housing 10 toward the bottom of the housing 10 (the negative Z-axis direction in the figure) until the discharge port 22a is positioned at the print start position Ps. Here, the print start position Ps is within the first support material 1.

[0028] After positioning the discharge port 22a at the print start position Ps, the control device 40 controls the drive device 30 to move the discharge port 22a in a lifting direction D3. The lifting direction D3 is the direction from the first support material 1 toward the second support material 2, which is the positive direction of the Z axis in the drawing.

[0029] The control device 40 controls the bioprinter 20 and the driving device 30 so that the bioink is ejected from the ejection port 22a while moving the ejection port 22a in the lifting direction D3, thereby printing the bioink linearly within the substrate.

[0030] The control device 40 controls the pump 24 to begin applying pressure to the bioink in the tube 22 in the discharge direction D1 after the discharge port 22a has been inserted into the substrate and before the discharge port 22a begins moving from the print start position Ps in the lifting direction D3.

[0031] Figure 4 is a schematic diagram showing an example of successful printing. Figure 5 is a schematic diagram showing an example of unsuccessful printing. For example, as shown in Figure 5, if pressure is applied to the bioink in the discharge direction D1 at the same time that the discharge port 22a starts moving in the lifting direction D3 from the print start position Ps, printing of the bioink Bi may not start from the print start position Ps due to reasons such as the substrate (first support material 1, second support material 2, and support material 3) getting stuck in the syringe when the tube 22 is inserted.

[0032] In particular, the bioprinter 20 according to this embodiment has a void Ga formed within the pump housing 242. Because the compressibility of air is greater than that of the substrate, the void Ga compresses the air within the void Ga when the nozzle 224 is inserted into the substrate, allowing the substrate to more easily enter the nozzle 224. Furthermore, because the compressibility of air is greater than that of the bioink, when the bioink is ejected, the air within the void Ga is compressed when the piston 244 begins to move in the ejection direction D1, and the pressure is then transmitted to the bioink within the nozzle 224. Therefore, a time lag occurs between when pressure begins to be applied to the bioink in the ejection direction D1 and when the bioink is actually ejected from the ejection port 22a. If pressure is applied at the start of movement in the lifting direction D3, this time lag can cause problems, such as printing not starting from the desired print start position Ps. As a result, the printed bioink line may not be the desired length, potentially resulting in failure to obtain cell tissue of the desired length.

[0033] Furthermore, if printing does not start from the desired print start position Ps, and printing starts from within support material 3 rather than within first support material 1, one end of the bioink cannot be supported by first support material 1, and the desired growth environment cannot be provided for skeletal muscle cells, which are cells within the bioink.

[0034] In this embodiment, pressure is applied to the bioink in the ejection direction D1 before the ejection outlet 22a starts moving from the print start position Ps in the lifting direction D3, so that printing starts from the desired print start position Ps, as shown in Figure 4, and the bioink can be printed to the desired length.

[0035] Furthermore, since printing starts from the print start position Ps, one end of the printed linear bio-ink can be supported by the first support material 1.

[0036] The timing at which the control device 40 starts applying pressure in the discharge direction D1 may be any timing as long as it is before the start of movement in the lifting direction D3, and may be, for example, while the discharge port 22a is moving toward the bottom inside the housing 10. Furthermore, the control device 40 may cause the discharge port 22a to remain at the print start position Ps for a predetermined period after the discharge port 22a reaches the print start position Ps, and may start applying pressure in the discharge direction D1 while the discharge port 22a is remaining at the print start position Ps.

[0037] [Control method] 6 is a flowchart showing the processing executed by the control device. Note that the processing shown in FIG. 6 will be explained assuming that it is executed after the bio-ink is contained in the nozzle 224.

[0038] In S100, the control device 40 controls the drive device 30 to move the bioprinter 20 so that the discharge port 22a is at the print start position Ps.

[0039] In S102, the control device 40 determines whether the ejection opening 22a has reached the print start position Ps. For example, the drive device 30 transmits the drive amount in the horizontal direction and the vertical direction to the control device 40. Based on the information received from the drive device 30, the control device 40 determines whether the ejection opening 22a has reached the print start position Ps.

[0040] If it is determined that the discharge port 22a has reached the print start position Ps (YES in S102), the control device 40 instructs the drive device 30 to stop moving the bioprinter 20 in S104. The control device 40 may notify the drive device 30 of the drive amount and determine that the discharge port 22a has reached the print start position Ps by receiving a notification from the drive device 30 that the movement by the notified drive amount has been completed.

[0041] In S106, the control device 40 controls the bioprinter 20 to start the ejection operation. Specifically, the control device 40 commands the bioprinter 20 to start applying pressure to the bioink in the ejection direction D1. In response to this command, the bioprinter 20 starts moving the piston 244 in the ejection direction D1.

[0042] In S108, the control device 40 determines whether a predetermined period of time has elapsed since the start of the discharge operation. The predetermined period of time is set appropriately by a person skilled in the art depending on the capacity of the pump 24, the hardness of the base material, and the inner diameter of the nozzle 224. As an example, when the pump capacity is 2 μL / s, the base material is gelatin, and the inner diameter of the nozzle 224 is 1.6 mm, the predetermined period is set to 2 seconds.

[0043] If it is determined that a predetermined period of time has elapsed since the start of the discharging operation (YES in S108), the control device 40 controls the drive device 30 in S110 to move the bioprinter 20 in the lifting direction D3 at a predetermined speed. At this time, since the bioprinter 20 has received a command to start the discharging operation in S106, pressure continues to be applied to the bioink in the discharging direction D1 while the bioprinter 20 is being lifted, and the bioink is discharged from the discharge port 22a.

[0044] In S112, the control device 40 determines whether the discharge port 22a has reached the print end position. The print end position is, for example, in the second support material 2. For example, the control device 40 determines whether the discharge port 22a has reached the print end position based on information on the drive amount received from the drive device 30.

[0045] If it is determined that the discharge port 22a has reached the printing end position (YES in S112), the control device 40 commands the drive device 30 to stop the movement of the bioprinter 20 in S114, and commands the bioprinter 20 to stop the discharge operation in S116, thereby ending the processing.

[0046] After this process is completed, the control device 40 may perform a process of sucking bio-ink into the tube 22 in order to start the next printing operation. After that, the process shown in FIG. 6 may be started again.

[0047] As described above, the culture device 100 according to this embodiment can reliably start printing from the print start position Ps by applying pressure for a predetermined period of time while the discharge port 22a remains at the print start position Ps and then starting the lifting operation. If pressure is started while the discharge port 22a is moving toward the bottom inside the housing 10, printing may start before the discharge port 22a reaches the print start position Ps, which may increase the amount of bioink consumed. On the other hand, the culture device 100 according to this embodiment starts applying pressure after the discharge port 22a is positioned at the print start position Ps, thereby preventing unnecessary consumption of bioink.

[0048] In the above embodiment, the bioprinter 20 is provided with one tube 22 (nozzle 224), but it may be a multi-nozzle dispenser provided with a plurality of tubes 22 (nozzles 224).

[0049] [Aspect] It will be understood by those skilled in the art that the above-described embodiments are specific examples of the following aspects.

[0050] (Item 1) A culture device according to one embodiment is a culture device for bioprinting, which prints a line of bioink containing cells to obtain a line of cellular tissue. The culture device includes a bioprinter that ejects the bioink to print the bioink in a line, a housing configured to accommodate a substrate that supports the bioink, a drive device that moves the bioprinter and the housing relative to one another, and a control device. The bioprinter includes a tube that accommodates the bioink and a pump that applies pressure to the bioink in a direction toward the tube's outlet. The control device positions the outlet at a print start position within the housing that accommodates the substrate, and then controls the drive device to move the outlet in a first direction within the substrate within the housing. The control device controls the pump to begin applying pressure to the bioink in a direction toward the outlet after the outlet is inserted into the substrate and before the outlet begins moving in the first direction from the print start position.

[0051] According to the culture device described in paragraph 1, the bioink can be printed to the desired length by starting to apply pressure to the bioink in a direction toward the discharge port before the discharge port starts moving in the first direction from the printing start position.

[0052] (Item 2) In the culture device described in item 1, the control device positions the discharge port at a print start position and then controls the drive device to keep the discharge port at the print start position. After the control device positions the discharge port at the print start position and before the discharge port starts moving in a first direction, the control device controls the pump to start applying pressure to the bio-ink in a direction toward the discharge port. The control device controls the drive device to move the discharge port in the first direction after a predetermined period has elapsed since the pump started applying pressure to the bio-ink in a direction toward the discharge port.

[0053] According to the culture device described in paragraph 2, by applying pressure for a predetermined period while the discharge port is kept at the print start position and then starting movement in the first direction, printing can be reliably started from the print start position. In addition, because pressure is applied only after the discharge port is positioned at the print start position, wasteful consumption of bioink can be prevented.

[0054] (Item 3) In the culture device described in item 1 or 2, the substrate includes a first support material that supports one end of the printed linear bioink, a second support material that supports the other end of the linear bioink, and a retaining material that is disposed between the first support material and the second support material. The first support material, the retaining material, and the second support material are housed in the housing in this order from the bottom side of the housing. The printing start position is within the first support material. The first direction is the direction from the first support material toward the second support material.

[0055] According to the culture device described in the third aspect, printing starts from within the first support material, so one end of the printed linear bio-ink can be supported by the first support material.

[0056] (Item 4) In the culture device according to any one of items 1 to 3, the tube is configured to be detachable from the pump. The pump includes a pump housing having a connection port for the tube and a piston configured to be able to reciprocate within the pump housing. The pump housing is configured so that, when the piston moves to a position closest to the connection port during reciprocating motion, a gap of a predetermined volume is formed between the open end of the connection port and the piston.

[0057] According to the culture device described in paragraph 4, the tube is detachable from the pump and can be easily replaced. This makes the tube disposable and the pump reusable, which is hygienic and economical. In addition, a gap is formed inside the pump housing, which prevents bioink from seeping into the pump housing when it is sucked in. This eliminates the need to clean and sterilize the inside of the pump when the bioprinter is used repeatedly.

[0058] (Item 5) A printing method according to one embodiment is a method for printing bioink containing cells that will become cellular tissue when cultured into a substrate that supports the bioink. The printing method includes the steps of positioning an outlet of a bioprinter for printing the bioink in a line at a print start position within the substrate, moving the outlet in a first direction within the substrate from the print start position, and, after the outlet is inserted into the substrate but before the outlet starts moving in the first direction, starting to apply pressure to the bioink in the bioprinter in a direction toward the outlet.

[0059] According to the culture device described in paragraph 5, the bioink can be printed to the desired length by starting to apply pressure to the bioink in a direction toward the discharge port before the discharge port starts moving in the first direction from the printing start position.

[0060] (Item 6) A program according to one aspect is a program that causes a computer to execute the printing method described in item 5.

[0061] (Item 7) A recording medium according to one aspect is a computer-readable recording medium on which the program according to item 6 is recorded.

[0062] 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]

[0063] 1 first support material, 2 second support material, 3 support material, 10 housing, 20 bioprinter, 22 tube, 22a outlet, 24 pump, 30 drive device, 40 control device, 42 processor, 44 memory, 50 input device, 60 display device, 100 culture device, 222, 242a connection port, 224 nozzle, 242 pump housing, 242b open end, 242c storage section, 242d tip, 244 piston, Bi bioink, D1 outlet direction, D2 suction direction, D3 pull-up direction, Ga void, Ps print start position.

Claims

1. A bioprinting culture device for obtaining linear cell tissue by linearly printing a bioink containing cells, comprising: a bioprinter that ejects the bioink and prints the bioink in a linear pattern; a housing configured to accommodate a substrate that supports the bioink; a drive device that moves the bioprinter and the housing relative to one another; a control device; The bioprinter includes: a tube containing the bioink; a pump for applying pressure to the bioink in a direction toward the outlet of the tube, The control device After positioning the ejection opening at a print start position within the housing in which the substrate is accommodated, the drive device is controlled so that the ejection opening moves within the substrate within the housing in a first direction; A culture device that controls the pump to begin applying pressure to the bioink in a direction toward the outlet after the outlet is inserted into the substrate and before the outlet starts moving in the first direction from the print start position.

2. The control device After the ejection opening is positioned at the print start position, the drive device is controlled so that the ejection opening remains at the print start position; controlling the pump to start applying pressure to the bio-ink in a direction toward the discharge port after the discharge port is positioned at the print start position and before the discharge port starts moving in the first direction; The culture device of claim 1 , wherein the drive device is controlled so that the outlet moves in the first direction after a predetermined period of time has elapsed since the pump began to apply pressure to the bioink in a direction toward the outlet.

3. The substrate is a first support material supporting one end of the printed linear bioink, a second support material supporting the other end of the linear bioink, and a retaining material disposed between the first support material and the second support material; the first support material, the holding material, and the second support material are housed in the housing in this order from the bottom side of the housing; the start print location is within the first support; The culture device according to claim 1 or 2, wherein the first direction is a direction from the first support material toward the second support material.

4. The tube is configured to be detachable from the pump, The pump a pump housing having a connection port with the tube; and a piston configured to be capable of reciprocating within the pump housing, The culture device according to claim 1 or 2, wherein the pump housing is configured so that when the piston moves to a position closest to the connection port during reciprocating motion, a gap of a predetermined volume is formed between the open end of the connection port and the piston.

5. 1. A method for printing a bioink containing cells that can be cultured to form a cellular tissue into a substrate that supports the bioink, comprising: Positioning an outlet of a bioprinter for printing the bioink linearly at a print start position within the substrate; moving the outlet in a first direction within the substrate from the print start position; and after the outlet is inserted into the substrate and before the outlet starts moving in the first direction, starting to apply pressure to the bioink in the bioprinter in a direction toward the outlet.

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