Bioprinter using a bio-material freeze-hardening method and freeze-hardening method thereof

Abstract

A bioprinter applying a biological material freeze-hardening method includes a plurality of dispensers configured to discharge a biological material within a print chamber, a print plate module disposed below the dispensers such that the discharged biological material is placed thereon, a cooling module including one or more Peltier elements configured to cool the print plate module and a water tank disposed on a heating surface side of the Peltier elements and having a coolant flow path formed therein, a coolant supply module configured to supply a coolant to the cooling module, a temperature sensor disposed in the cooling module to calculate a temperature of a print surface of the print plate module, a print controller configured to control the dispensers to print an output, and an output hardening controller configured to, after the print controller completes printing of the output, receive a detection value of the temperature sensor and control the cooling module and the coolant supply module to freeze-harden the output.

Description

[0001] Cross-references to related applications

[0002] This application claims priority and benefit to Korean Patent Application No. 10-2021-0171689, filed on December 3, 2021, the disclosure of which is incorporated herein by reference in its entirety. Technical Field

[0003] This disclosure relates to a bioprinter that uses a cryo-curing method for biomaterials and the cryo-curing method thereof, which is capable of cryo-curing biomaterials printed on a printing surface by the bioprinter to maintain the form of the discharged biomaterials. Background Technology

[0004] In recent years, with the increasing number of research projects in tissue engineering and regenerative medicine, as well as the demand for customized medical services, research on 3D printers using biomaterials has been actively carried out.

[0005] Known 3D printers consist of a frame that forms the X, Y, and Z axes, and nozzles for ejecting biological material. In conventional printers, a liquid dispenser is filled with a viscous fluid biological material, such as collagen or gelatin. The fluid biological material filling the liquid dispenser is ejected through the nozzles onto the printing plate.

[0006] However, when the biomaterial is in a solid rather than fluid state, the nozzle must be heated to a high temperature to discharge the flowable biomaterial. Therefore, in order to use both fluid and solid biomaterials together, the process of heating and cooling the nozzle is repeated, thus overloading the nozzle.

[0007] To address this issue, Korean Patent No. 10-1828345 discloses a 3D bioprinter that includes a first dispenser and a second dispenser. The first dispenser is configured to melt and dispense solid biomaterials for forming scaffolds and other structures, and the second dispenser is configured to print fluid biomaterials.

[0008] The aforementioned registered patent discloses a method for hybrid laminating solid and liquid biomaterials into a single structure. One example of hybrid lamination includes a method in which a biomaterial, such as polycaprolactone (PCL), is used to form a scaffold on a printing surface, and then liquid bioink is printed to fill the scaffold.

[0009] However, liquid bio-inks have high fluidity, making their form easily changeable. For biomaterials to function properly in a living organism, they should be created in a form suitable for a specific part of the organism before being applied. When the form of the biomaterial extruded onto the printing surface is easily altered, it is difficult to apply the biomaterial to a living organism.

[0010] Accordingly, there is a need for an apparatus that allows a bio-material discharged from a fluid bio-material dispenser in a dispenser of a bioprinter to be applied to a living body while maintaining the form of the bio-material on a printing surface.

[0011] [Related Art Documents]

[0012] [Patent Documents]

[0013] (Patent Document 1) Korean Patent No. 10-1828345 (Publication Date: March 29, 2018) SUMMARY

[0014] The present disclosure relates to a bioprinter applying a bio-material freeze-hardening method and a freeze-hardening method thereof, which allows a bio-material discharged from a fluid bio-material dispenser in a dispenser of a bioprinter to be immediately hardened on a printing surface and applied to a living body while maintaining its shape.

[0015] A first aspect of the present disclosure relates to a bioprinter applying a bio-material freeze-hardening method. The bioprinter can include a plurality of dispensers configured to discharge a bio-material within a printing chamber, a printing plate module disposed below the dispensers such that the discharged bio-material is placed thereon, a cooling module including one or more Peltier elements configured to cool the printing plate module and a water tank disposed on a heating surface side of the Peltier elements and having a coolant flow path formed therein to circulate a coolant to dissipate heat generated by the Peltier elements, a coolant supply module configured to supply the coolant to the cooling module, a temperature sensor disposed in the cooling module to calculate a temperature of a printing surface of the printing plate module, a print controller configured to control the operation of the dispensers to print an output, and an output hardening controller configured to, after the print controller completes the printing of the output, receive a detection value from the temperature sensor and control the cooling module and the coolant supply module to freeze-harden the output.

[0016] According to an embodiment of the present disclosure, the printing plate module can include a cooling bed disposed on and cooled by the Peltier elements and a substrate detachably mounted on the cooling bed, wherein a biocompatible film can be detachably attached to an upper surface of the substrate due to adhesion, and the discharged bio-material is placed on a surface of the biocompatible film.

[0017] According to an embodiment of the disclosure, the plurality of dispensers can include a first dispenser configured to print a scaffold that defines an outer boundary of the output, and a second dispenser configured to print a fluid bio-material in the scaffold, wherein the scaffold can be fused and fixed to the membrane as the membrane is partially melted due to the bio-material printed by the first dispenser, and the fluid bio-material printed by the second dispenser can be designed not to leak through an interface between the scaffold and the membrane.

[0018] According to an embodiment of the disclosure, the output formed by the scaffold and the fluid bio-material and freeze-hardened can be a patch implanted in an affected area or a traumatic defect of a tissue or an organ of a mammal or a human.

[0019] According to an embodiment of the disclosure, the affected area can be an affected area in which a diabetic foot ulcer or cartilage abrasion has occurred or a traumatic defect due to a burn or a cut has occurred.

[0020] According to an embodiment of the disclosure, the first fixing surface and the second fixing surface can be disposed at positions facing each other on the cooling bed, the first fixing surface and the second fixing surface are configured to fix the mounted substrate, and a handle can be formed at the substrate, the handle extending to a position spaced apart from the cooling bed.

[0021] According to an embodiment of the disclosure, the second fixing surface can be formed in a direction toward a door of the print chamber, a fitting space can be formed at the second fixing surface, and the handle is fitted into the fitting space.

[0022] According to an embodiment of the disclosure, a thermal grease layer can be formed between an upper surface of the Peltier element and a lower surface of the cooling bed and between a lower surface of the Peltier element and an upper surface of the water tank.

[0023] According to an embodiment of the disclosure, a Peltier support can be formed between the Peltier element and the water tank, and a plurality of grooves spaced apart from each other can be formed in an upper surface of the Peltier support, the Peltier element being fitted in the grooves.

[0024] According to an embodiment of the disclosure, the coolant supply module can include a coolant tank configured to store a coolant and receive the circulated coolant from the water tank, a coolant pump connected to the coolant tank, a heat sink having one side connected to the coolant pump and the other side connected to the water tank and having a coolant circulation flow path formed therein, and a heat sink fan configured to dissipate heat generated from the heat sink.

[0025] According to an embodiment of the disclosure, the chamber exterior space can be formed separately from an interior space of the print chamber, a coolant supply module is installed in the chamber exterior space, and the chamber exterior space can include at least one duct fan configured to discharge heat generated in the chamber exterior space to the outside of the bioprinter, an outside air suction port through which outside air is introduced into the chamber exterior space, and a coolant refill door formed to be openable and closable to allow the coolant to be supplied from the outside to the coolant tank and formed above a coolant inlet of the coolant tank.

[0026] According to an embodiment of the disclosure, the output hardening controller can operate the coolant pump, the radiator fan, and the duct fan while operating the Peltier element, can stop the operation of the Peltier element in response to the output being frozen and hardened due to the temperature of the print surface being cooled to a predetermined temperature, and can additionally operate the coolant pump, the radiator fan, and the duct fan for a predetermined amount of time after stopping the operation of the Peltier element in order to remove residual heat from the Peltier element.

[0027] According to an embodiment of the disclosure, the output hardening controller can control the operation of the Peltier element so that the temperature of the print surface reaches a predetermined temperature in the range of -1℃ to -20℃.

[0028] A second aspect of the disclosure relates to a method of freeze-hardening a biomaterial of a bioprinter. The method is a method of freeze-hardening a biomaterial of a bioprinter including a plurality of dispensers, a print plate module, a cooling module including a Peltier element, a temperature sensor disposed in the cooling module, a coolant supply module including a coolant tank, a coolant pump, a radiator, and a radiator fan, a duct fan configured to discharge heat to the outside, a print controller, and an output hardening controller, the method can include: (a) an operation of discharging a biomaterial in a print chamber to form an output by the plurality of dispensers by the print controller; (b) an operation of operating the coolant pump, the radiator fan, and the duct fan while operating the Peltier element; and (c) an operation of stopping the operation of the Peltier element in response to the output being freeze-hardened due to the temperature of a print surface of the print plate module being cooled to a predetermined temperature, the temperature of the print surface of the print plate module being calculated based on a value detected by the temperature sensor.

[0029] According to embodiments of the present disclosure, the plurality of dispensers can include a first dispenser and a second dispenser, the biocompatible membrane can be detachably attached to the upper surface of the print plate module due to adhesion, step (a) can include: (a-1) an operation of printing a scaffold of the output by the first dispenser, the scaffold defining an outer boundary of the output; (a-2) an operation of printing a fluid biomaterial in the scaffold by the second dispenser; and (a-3) an operation of fusing the scaffold to the membrane when the membrane is partially melted due to the biomaterial printed by the first dispenser, wherein the fluid biomaterial printed by the second dispenser can be designed not to leak through the interface between the scaffold and the membrane.

[0030] According to embodiments of the present disclosure, the method can further include, after (c), (d) additionally operating the coolant pump, the heat sink fan, and the duct fan for a predetermined amount of time after stopping the operation of the Peltier element, so as to remove residual heat from the Peltier element. BRIEF DESCRIPTION OF DRAWINGS

[0031] The above and other objects, features and advantages of the present disclosure will become more apparent to one of ordinary skill in the art by describing in detail exemplary embodiments thereof with reference to the attached drawings in which:

[0032] Figure 1 is a view illustrating a bioprinter to which a freeze hardening method according to the present disclosure is applied;

[0033] Figure 2 is a block diagram illustrating each component of a water tank to provide coolant to the Figure 1

[0034] Figure 3 is a side view illustrating each component of a print plate module of the Figure 1

[0035] Figure 4 is a plan view illustrating an arrangement of a Peltier element of the Figure 2

[0036] Figure 5 is a plan view illustrating an arrangement of a water tank of the Figure 2

[0037] Figure 6 is a view illustrating a coolant circulation path between the water tank and the coolant providing module of the Figure 2

[0038] Figure 7 is a view illustrating a coolant tank and a coolant pump in the coolant providing module of the Figure 2

[0039] Figure 8 is a view illustrating a coolant tank and a coolant pump in the coolant providing module of the Figure 1 ​​​​​​a view of an external space of a chamber outside of a bioprinter of

[0040] Figure 9 is a view showing a cooling bed of Figure 1 a bioprinter of

[0041] Figure 10 is a view showing a Peltier element and a temperature sensor disposed between the Peltier element of Figure 3

[0042] Figure 11 is a perspective view showing a state before a film and a substrate are coupled to a cooling bed of Figure 2

[0043] Figure 12 is a perspective view showing a state when a complex of a film and a substrate is being coupled to a cooling bed of Figure 11

[0044] Figure 13 is a perspective view showing a state after a complex of a film and a substrate is coupled to a cooling bed of Figure 11 DETAILED DESCRIPTION

[0045] Hereinafter, details for implementing the present disclosure will be described with reference to the accompanying drawings. In describing the present disclosure, when a detailed description of related known functions is deemed to make it possible to unnecessarily obscure the gist of the present disclosure for those of ordinary skill in the art, a detailed description thereof will be omitted.

[0046] Figure 1 is a view showing a bioprinter to which a freeze hardening method according to the present disclosure is applied. Figure 2 is a block diagram showing each component for providing a coolant to a water tank of Figure 1

[0047] With reference to Figure 1 and Figure 2 , a bioprinter to which a freeze hardening method according to the present disclosure is applied includes a print chamber 1 and a door 2 formed as openable and closable on the print chamber 1, and printing of a biomaterial is performed in an internal space closed by the door 2.

[0048] The bioprinter includes a dispenser 10, a print plate module 20, a cooling module 30, a coolant providing module 40, a temperature sensor 50, a print controller 61, and an output hardening controller 62.

[0049] ​​​​​The dispenser 10 is provided as one or more dispensers 10 and is formed to discharge a bio material through a nozzle and is movable upward, downward, leftward, and rightward. The dispenser 10 can be constituted of a first discharge module and a second discharge module, the first discharge module being detachably mounted on the second discharge module. The dispenser 10 can be constituted of any one of a first dispenser 11 configured to melt and discharge a solid bio material and a second dispenser 12 configured to discharge a liquid bio ink.

[0050] A bio material holder 13 having the bio material wound thereon is provided at an upper side of the first dispenser 11. The bio material can be made of a bio material to which a granular melting method using pneumatic pressure is applied, or can be made of a polycaprolactone (PCL) bio material. The first dispenser 11 prints a scaffold that defines an outer boundary of an output. The bio material wound on the bio material holder 13 is supplied to the first dispenser 11, heated to a predetermined temperature in the first dispenser 11, and then discharged through a nozzle. The first dispenser 11 is constituted of a 1-1 discharge module 11a and a 1-2 discharge module 11b. The nozzle is mounted on an end portion of the 1-1 discharge module 11a, and the 1-1 discharge module 11a is detachably mounted on the 1-2 discharge module 11b. A cooling fan 14 configured to cool the bio material discharged from the first dispenser 11 can be provided at a lateral portion of the nozzle of the first dispenser 11.

[0051] The second dispenser 12 discharges a liquid bio ink. The second dispenser 12 prints a fluid bio material in a scaffold discharged from the first dispenser 11. The second dispenser 12 is constituted of a 2-1 discharge module 12a and a 2-2 discharge module 12b. The 2-1 discharge module 12a is formed in the shape of a syringe and has a nozzle mounted on an end portion. The 2-2 discharge module 12b includes a fixer 12b-1 configured to fix the syringe and a presser 12b-2 configured to receive power from a pneumatic pressure provider or a stepping motor (not shown) and press a plunger rod of the syringe to discharge a bio ink in the syringe.

[0052] Figure 3 is a side view showing each component of the print plate module of Figure 1 .

[0053] Referring to Figure 3 , the print plate module 20 is provided below the dispenser 10, has a print surface 20a on which a discharged bio material is placed, and is formed to be fixed or formed to be movable on a horizontal plane.

[0054] The bio-material discharged from the dispenser 10 is placed on the printing surface 20a. The print bed module 20 can include a film 21, a base plate 22, and a cooling bed 23 from top to bottom. The film 21 is detachably attached to the upper surface of the base plate 22, and in this case, the upper surface of the film 21 is the printing surface 20a. The film 21 can be made of a biocompatible material made of polyurethane. The base plate 22 is detachably mounted on the cooling bed 23. The film 21 easily adheres to the molten polymer material and prevents the liquid bio-ink from leaking out of the scaffold. When the film 21 is partially melted due to the bio-material printed by the first dispenser 11, the scaffold is fused and fixed to the film 21. Therefore, the fluid bio-material printed by the second dispenser 12 does not leak through the interface between the scaffold and the film 21. The output formed by the scaffold and the fluid bio-material and cryohardened can be configured as a patch to be implanted in an affected area or a traumatic defect of a tissue or an organ of a mammal or a human. In addition, the affected area can be an affected area in which a diabetic foot ulcer or cartilage abrasion has occurred, or an affected area in which a traumatic defect has occurred due to a burn or a cut.

[0055] Figure 4 is a plan view showing an arrangement of Peltier elements of Figure 2 . Figure 5 is a plan view showing an arrangement of a water tank of Figure 2 .

[0056] Referring to Figure 4 and Figure 5 , the cooling module 30 includes one or more Peltier elements 31 configured to cool the print bed module 20 and a water tank 32 disposed on a heating surface side of the Peltier elements 31 and having a coolant flow path formed therein to circulate a coolant to dissipate heat generated by the Peltier elements 31. A plurality of Peltier elements 31, for example, four Peltier elements 31, can be arranged in parallel. The Peltier elements 31 are disposed above and supported by a Peltier support 32c. The Peltier support 32c is formed as an upper surface of the water tank 32, and a plurality of grooves into which the Peltier elements 31 are fitted are formed in the upper surface of the Peltier support 32c. The Peltier elements 31 are spaced apart from each other, and a region filled with air is formed between the Peltier elements 31.

[0057] The water tank 32 can be formed of brass and / or aluminum having high thermal conductivity. A thermal grease layer 33 can be formed between the upper surface of the Peltier element 31 and the lower surface of the cooling bed 23 and between the lower surface of the Peltier element 31 and the upper surface of the water tank 32. The thermal grease layer 33 can be formed of aluminum metal particles, zinc oxide (ZnO) (semiconductor particles), and / or silicon oil. A water inlet 32a through which coolant flows in is formed at one side of the coolant flow path of the water tank 32, and a water outlet 32b through which coolant flows out is formed at the other side of the coolant flow path of the water tank 32. The water inlet 32a and the water outlet 32b pass through a connector block 35 coupled to the lower portion of the water tank 32 and are connected to a coolant flow pipe extending from the coolant supply module 40.

[0058] Figure 6 is a view illustrating a coolant circulation path between the water tank of Figure 2 and the coolant supply module. Figure 7 is a view illustrating a coolant tank and a coolant pump in the coolant supply module of Figure 2 .

[0059] Referring to Figure 6 and Figure 7 , the coolant supply module 40 is designed to supply coolant to the cooling module 30 and includes a coolant tank 41, a coolant pump 42, a radiator 43, and a radiator fan 44.

[0060] The coolant tank 41 stores coolant and receives circulated coolant from the water tank 32. Coolant inlets 41a and 41b are formed in the coolant tank 41, the coolant inlet 41a is configured to receive coolant from the outside, and the coolant flows in through the inlet 41b from the water tank 32. The coolant pump 42 is connected to the coolant tank 41 and provides power to the coolant received from the coolant tank. A connection port 42a is formed in the coolant pump 42, the connection port 42a is connected to the coolant tank 41, and the coolant flows out through an outlet 42b. The coolant flowing out through the outlet 42b is supplied to the radiator 43. One side of the radiator 43 is connected to the coolant pump 42, and the other side is connected to the water tank 32. A coolant circulation flow path is formed in the radiator 43, and the coolant circulates along the coolant circulation flow path in the radiator 43. The radiator fan 44 is disposed adjacent to the radiator 43 to dissipate heat generated from the radiator 43.

[0061] Figure 8 is a view illustrating a chamber external space in which a controller and a coolant supply module are disposed in the bioprinter of Figure 1 . Figure 9 is a view illustrating an external air suction port and a coolant refill door installed in the chamber external space of the bioprinter of Figure 1 .

[0062] Referring to Figure 8 and Figure 9 , a bioprinter according to the present disclosure can include a print chamber 1a having an internal space in which printing is performed, and a chamber exterior space 1b separated from the print chamber 1a by a partition 3. The chamber exterior space 1b is provided at an upper side of the print chamber 1a. A coolant supply module 40, a print controller 61, and an output hardening controller 62 are provided in the chamber exterior space 1b.

[0063] A duct fan 71, an external air suction port 72, and a coolant refill door 73 can be installed in the chamber exterior space 1b. The duct fan 71 discharges heat generated in the chamber exterior space 1b to the outside of the bioprinter. The duct fan 71 can be installed as a plurality of duct fans 71 on the side wall of the chamber exterior space 1b. The external air suction port 72 is a passage that introduces external air into the chamber exterior space 1b. The coolant refill door 73 is formed to be openable and closable to supply coolant to the coolant tank 41, and is formed above the coolant inlet 41a of the coolant tank 41. The external air suction port 72 and the coolant refill door 73 can be installed on the upper surface of the chamber exterior space 1b. The external air suction port 72 can be provided as a plurality of external air suction ports 72.

[0064] Figure 10 is a view showing Figure 3 a Peltier element and a temperature sensor provided between the Peltier elements.

[0065] Referring to Figure 10 , a temperature sensor 50 is provided in the cooling module 30 to calculate the temperature of the print surface 20a of the print plate module 20. For example, the temperature sensor 50 can be provided between the Peltier elements 31, and can be located at the center of the arrangement of the Peltier elements 31. Using a pre-calculated table reflecting the difference in height between the temperature sensor 50 and the print surface 20a, the temperature of the print surface 20a can be calculated from the value detected by the temperature sensor 50.

[0066] The print controller 61 is configured to control the operation of the dispenser 10 to print an output. The output hardening controller 62 is configured to receive a detection value from the temperature sensor 50 after the print controller 61 completes the printing of the output, and to control the cooling module 30 and the coolant supply module 40 to freeze and harden the output. The output is cooled by the Peltier elements 31, so that the output is freeze-hardened. The output hardening controller 62 can control the operation of the Peltier elements 31, the coolant supply module 40, and the duct fan 71. The output hardening controller 62 can control the operation of the Peltier elements 31 so that the temperature of the print surface 20a reaches a predetermined temperature in the range of -1℃ to -20℃.

[0067] The output hardening controller 62 performs the method of hardening a biomaterial of a bio-printer according to the present disclosure as follows.

[0068] First, the output hardening controller 62 operates the coolant pump 42, the radiator fan 44, and the duct fan 71 while operating the Peltier element 31 (S10). The output hardening controller 62 receives a temperature value detected by the temperature sensor 50 and controls the Peltier element 31 using a proportional-integral-derivative (PID) method. The power of the Peltier element 31 is controlled by a solid state relay (SSR), and the power supply method is controlled by pulse width modulation (PWM). The output hardening controller 62 operates the Peltier element 31 by real-time PID control. Next, in response to the temperature detected by the temperature sensor 50 reaching a predetermined temperature in the range of -1℃ to -20℃, the output hardening controller 62 turns off the SSR to stop the operation of the Peltier element 31 (S20). The printing temperature is cooled to the predetermined temperature, and the freeze hardening of the output is completed. In response to the temperature detected by the temperature sensor 50 not reaching the predetermined temperature, the output hardening controller 62 turns on the SSR. Next, after stopping the operation of the Peltier element 31, the output hardening controller 62 additionally operates the coolant pump 42, the radiator fan 44, and the duct fan 71 for a predetermined amount of time (S30). The predetermined amount of time can be set to about 1 minute. In this way, malfunction of the Peltier element 31 can be prevented.

[0069] Figure 11 is a perspective view showing a state before a film and a substrate are coupled to a cooling bed of Figure 2 . Figure 12 is a perspective view showing a state when a composite of a film and a substrate is being coupled to a cooling bed of Figure 11 . Figure 13 is a perspective view showing a state after a composite of a film and a substrate is coupled to a cooling bed of Figure 11 .

[0070] Referring to Figures 11 to 13The complex of the film 21 and the substrate 22 is coupled to the cooling bed 23. The film 21 attached to the substrate 22 is detachably coupled to the cooling bed 23. A first fixing surface 23a and a second fixing surface 23b are provided at positions facing each other on the cooling bed 23, and are configured to fix the mounted substrate 22. A handle 22a extending to a position spaced apart from the cooling bed 23 is formed at the substrate 22. The second fixing surface 23b is formed in a direction toward a door of the printing chamber, and a fitting space 23c is formed on the second fixing surface 23b into which the handle 22a is fitted. The handle 22a of the other side of the substrate 22 is fitted into the fitting space 23c in a state in which one side of the substrate 22 is inclined while being in contact with the first fixing surface 23a. The other side of the substrate 22 is in contact with the second fixing surface 23b, and the substrate 22 is fixed to the cooling bed 23. After printing and cooling are completed, a worker can hold the handle 22a, lift the other side of the substrate 22, and then detach the other side of the substrate 22 from the cooling bed 23.

[0071] According to the present disclosure, a bio-material discharged from a fluid bio-material dispenser in a dispenser of a bioprinter can be immediately hardened on a printing surface and applied to a living body while maintaining its shape.

[0072] The scope of protection of the present disclosure is not limited by the description or representation of the embodiments explicitly described above. Furthermore, it should be noted that changes or replacements obvious in the art to which the present disclosure pertains cannot limit the scope of protection of the present disclosure.

Claims

1. A bioprinter applying a biological material freeze-hardening method, the bioprinter comprising: a plurality of dispensers configured to discharge a biological material within a print chamber; a print plate module disposed below the dispensers such that the discharged biological material is placed thereon; a cooling module including one or more Peltier elements configured to cool the print plate module and a water tank disposed on a heating surface side of the Peltier elements and having a coolant flow path formed therein to circulate a coolant to dissipate heat generated by the Peltier elements; a coolant supply module configured to supply the coolant to the cooling module; a temperature sensor disposed in the cooling module to calculate a temperature of a print surface of the print plate module; a print controller configured to control an operation of the dispensers to print an output; and an output hardening controller configured to, after the print controller completes printing of the output, receive a detection value from the temperature sensor and control the cooling module and the coolant supply module to freeze-harden the output.

2. The bioprinter of claim 1, wherein, The print plate module includes: a cooling bed disposed on and cooled by the Peltier elements; and a substrate detachably mounted on the cooling bed, and a biocompatible film detachably attached to an upper surface of the substrate due to adhesion, a surface of the biocompatible film having the discharged biological material placed thereon.

3. The bioprinter of claim 2, wherein, The plurality of dispensers includes: a first dispenser configured to print a scaffold defining an outer boundary of the output; and a second dispenser configured to print a fluid biological material in the scaffold, and when the film is partially melted due to the biological material printed by the first dispenser, the scaffold is fused and fixed to the film, and the fluid biological material printed by the second dispenser does not leak through an interface between the scaffold and the film.

4. The bioprinter of claim 3, wherein, The output formed by the scaffold and the fluid biological material and freeze-hardened is a patch implanted in an affected area of a skin or an organ of a mammal or a human.

5. The bioprinter of claim 4, wherein, The affected area is an affected area in which a diabetic foot ulcer or cartilage abrasion has occurred or a lesion or a traumatic defect has occurred in an organ. 6.The bioprinter of claim 2, wherein: a first fixing surface and a second fixing surface are disposed at positions facing each other on the cooling bed, the first fixing surface and the second fixing surface being configured to fix the mounted substrate; and a handle is formed at the substrate, the handle extending to a position spaced apart from the cooling bed.

7. The bioprinter of claim 6, wherein, The second fixing surface is formed in a direction toward a door of the print chamber, and a fitting space is formed at the second fixing surface, the handle fitting into the fitting space.

8. The bioprinter of claim 2, wherein, A heat grease layer is formed between the upper surface of the Peltier element and the lower surface of the cooling bed and between the lower surface of the Peltier element and the upper surface of the water tank. 9.The bioprinter of claim 1, wherein: a Peltier support is formed between the Peltier element and the water tank; and a plurality of grooves are formed in the upper surface of the Peltier support, the plurality of grooves being spaced apart from each other, the Peltier element being fitted in the plurality of grooves.

10. The bioprinter of claim 1, wherein, The coolant providing module includes: a coolant tank configured to store a coolant and receive a circulated coolant from the water tank; a coolant pump connected to the coolant tank; a heat sink having one side connected to the coolant pump and the other side connected to the water tank and having a coolant circulation flow path formed therein; and a heat sink fan configured to dissipate heat generated from the heat sink.

11. The bioprinter of claim 10, wherein, A chamber exterior space is formed separately from an interior space of the print chamber, the coolant providing module is installed in the chamber exterior space, and the chamber exterior space includes: at least one duct fan configured to exhaust heat generated in the chamber exterior space to the outside of the bioprinter; an outside air suction port through which outside air is introduced to the chamber exterior space; and a coolant refill door formed to be openable and closable to allow a coolant to be externally supplied to the coolant tank and formed above a coolant inlet of the coolant tank.

12. The bioprinter of claim 11, wherein, The output hardening controller: operates the coolant pump, the heat sink fan, and the duct fan while operating the Peltier element; stops the operation of the Peltier element in response to the output being frozen hardened due to the temperature of the print surface being cooled to a predetermined temperature; and after the operation of the Peltier element is stopped, additionally operates the coolant pump, the heat sink fan, and the duct fan for a predetermined amount of time in order to remove residual heat from the Peltier element. 13.The bioprinter of claim 1, wherein the output hardening controller controls the operation of the Peltier element such that the temperature of the print surface reaches a predetermined temperature in the range of -1℃ to -20℃. 14.A method of freeze-hardening a biomaterial of a bioprinter, the bioprinter including a plurality of dispensers, a print plate module, a cooling module including a Peltier element, a temperature sensor disposed in the cooling module, a coolant providing module including a coolant tank, a coolant pump, a heat sink, and a heat sink fan, a duct fan configured to exhaust heat to the outside, a print controller, and an output hardening controller, the method including: (a) an operation of discharging a biomaterial in a print chamber to form an output by the plurality of dispensers by the print controller; (b) an operation of operating the coolant pump, the heat sink fan, and the duct fan while operating the Peltier element; and (c) stopping the operation of the operation of the Peltier element in response to the output being frozen solid due to the temperature of the print surface of the print plate module being cooled to a predetermined temperature, the temperature of the print surface of the print plate module being calculated based on the value detected by the temperature sensor.

15. The method of claim 14, wherein the plurality of dispensers includes a first dispenser and a second dispenser, the biocompatible membrane being removably attached to the upper surface of the print plate module due to adhesion, and Step (a) includes: (a-1) an operation of printing a scaffold by the first dispenser, the scaffold defining an outer boundary of the output; (a-2) an operation of printing a fluid biological material in the scaffold by the second dispenser; and (a-3) an operation of fusing the scaffold to the membrane when the membrane is partially melted due to the biological material printed by the first dispenser, wherein the fluid biological material printed by the second dispenser does not leak through the interface between the scaffold and the membrane.

16. The method of claim 14, further comprising, after step (c), (d) an operation of additionally operating the coolant pump, the heat sink fan, and the duct fan for a predetermined amount of time to remove residual heat from the Peltier element after stopping the operation of the Peltier element.

Images