Six-degree-of-freedom robotic arm optimized for precise bio-ink loading and discharge control, control method thereof, and automated system and method for multi-precise dispensing and cell culture of viscoelastic bio-ink using same

Abstract

The present invention relates to the field of bioprinting and biotechnology, and to a system and method capable of improving quantitative dispensing accuracy through closed loop control using real-time weight feedback for hydrogel bioinks having viscoelasticity and shear thinning characteristics, and integrally performing multi-well dispensing and cell maintenance processes by robotic arm-based automation.

Description

A 6-degree-of-freedom robotic arm optimized for precise control of bioink loading and dispensing and a control method thereof, and an automated system and method for multi-precision dispensing of viscoelastic bioink and cell culture using the same

[0001] The present disclosure relates to a 6-degree-of-freedom robotic arm optimized for precise control of bioink loading and dispensing and a control method thereof, and more specifically, to a multi-precision dispensing and cell culture automation system and method of viscoelastic bioink using the same.

[0002] Currently, in the fields of tissue engineering and regenerative medicine, cell culture is primarily performed by researchers using instruments such as pipettes. Generally, this is carried out in an in vitro environment by injecting cell medium into well plates containing tissue engineering structures or by removing used medium and replacing it with fresh medium. This method involves relatively simple manual procedures and is widely used in cell culture research and experiments. In particular, the exchange of cell medium is considered an essential task to maintain cell viability and functionality during the cell culture process.

[0003] However, in the case of cell culture using cell-containing gels (bioinks), conventional methods present various problems. The process of encapsulating cells within the gel is complex, and because separating and handling the gel is difficult, the work process is cumbersome and time-consuming. Furthermore, the accuracy of this operation can vary depending on the researcher's skill level, and there is a risk of contamination during the process. In particular, when using a bioprinting pen manually, low precision leads to reduced accuracy and reproducibility, as well as an increased risk of contamination. This results in decreased work efficiency and negatively impacts the reliability of experimental results.

[0004] Furthermore, using high-viscosity hydrogel bioinks presents additional challenges. Because hydrogel bioinks exhibit shear-thinning properties, it is difficult to dispense them reproducibly in a consistent volume. This leads to irregular flow or clogging, which reduces operational consistency. In particular, when using gels containing expensive new drugs, low reproducibility can result in significant cost losses in in vitro, animal, and clinical trials, potentially leading to concerns regarding the reliability of the new drugs for consumers. These issues serve as major factors hindering the advancement of cell culture and bioprinting technologies.

[0005] The purpose of the present disclosure is to address the problems of reduced accuracy, lack of reproducibility, risk of contamination, and excessive labor burden arising from the loading, extrusion, and discharge processes of bioinks and the injection and exchange processes of cell culture media, which are performed relying on manual labor by researchers in the fields of cell culture and tissue engineering.

[0006] More specifically, the objective of the present disclosure is to provide a system and method that enables multi-position quantitative dispensing of bioink by stably and precisely controlling the loading and dispensing of viscoelastic bioink using a robotic arm, and improves the reproducibility and reliability of experiments by automating the cell culture process.

[0007] In addition, the objective of the present disclosure is to provide an automated cell culture platform applicable to various in vitro cell culture, tissue engineering, and drug evaluation experiments by enabling the precise injection or deposition of a hydrogel or bioink containing cells, drugs, or bioactive substances into each well of a cell culture plate.

[0008] The above and other objects and advantages of the present invention will become apparent from the following description describing preferred embodiments.

[0009] According to one embodiment of the present disclosure, a system for automating the multi-precision dispensing and cell maintenance processes of viscoelastic hydrogel bioink may be provided.

[0010] The above system may include a 6-degrees-of-freedom (DOF) robotic arm and an extrusion head module that is replaceably mounted on an end effector of the robotic arm, and the extrusion head module may be configured to load and discharge hydrogel bioink in at least one of a screw extrusion method, a pneumatic method, or a piston drive method.

[0011] In one embodiment, the system may further include a real-time weighing unit that measures the weight of the hydrogel bioink discharged by the extrusion head module in real time, and a closed-loop control unit that dynamically controls the extrusion amount of the extrusion head module until the hydrogel bioink reaches a set target dispensing amount based on weight information received from the real-time weighing unit. Through this, quantitative dispensing accuracy can be improved while compensating for rheological variability caused by the viscoelasticity and shear thinning characteristics of the hydrogel bioink.

[0012] In one embodiment, the system may include a central control unit that integrally controls the movement of the 6-degree-of-freedom robot arm and the closed-loop control unit, and the central control unit may be configured to control the movement path, discharge position, discharge sequence, and discharge pattern of the robot arm to dispense hydrogel bioink to a plurality of wells of a cell culture plate in a random or patterned manner.

[0013] In one embodiment, the system may further include an image scanning module including a webcam, and the central control unit may recognize position information of a dispensing target using image information obtained from the image scanning module and generate a dispensing position model of the 6-degree-of-freedom robot arm based on the position information. At this time, the recognition of the position information and the generation of the dispensing position model may be performed by a learning-based or rule-based control method.

[0014] In one embodiment, in addition to the extrusion head module, the end effector of the 6-degree-of-freedom robot arm may be replaceably equipped with a cell maintenance attachment for automatically sucking and removing used cell medium and injecting fresh cell medium in a set volume. The cell maintenance attachment may include a fluid control system comprising a syringe, a tube, a vacuum pump, a suction pump, a solenoid valve, and a control relay.

[0015] In one embodiment, the system may be placed in an environment chamber where temperature, humidity, and gas composition are controlled, and the 6-degree-of-freedom robotic arm may be configured to perform a cell culture process and a bioink dispensing process inside the environment chamber.

[0016] Additionally, according to one embodiment of the present disclosure, a method for multi-precision dispensing of viscoelastic hydrogel bioink using the system may be provided. The method may include the steps of: setting a target dispensing amount and converting it into a target weight; dispensing the hydrogel bioink to a target position using an extrusion head module mounted on the 6-degree-of-freedom robotic arm; measuring the weight of the hydrogel bioink in real time during dispensing; feedback controlling the dispensing amount of the extrusion head module until the target weight is reached based on the measured weight information; and moving to the next dispensing position or performing a cell medium exchange process after the target weight is achieved.

[0017] The present invention is a method shown as a model example in the detailed example provided below, but is not limited to the values ​​and processes shown below.

[0018] According to one embodiment of the present disclosure, by extruding hydrogel bioink using an extrusion head module that is replaceably mounted on a robotic arm, precision and reproducibility can be increased during the mixing of cells and gel and cell culture processes. According to one embodiment of the present disclosure, by dynamically controlling the dispensing amount of hydrogel bioink through a real-time metering unit and a closed-loop control unit, the accuracy of the dispensing process can be improved and the risk of contamination minimized.

[0019] The present invention controls a special extrusion motion based on two screw shapes mounted on a 6-axis robotic arm. This enables hydrogel mixing and extrusion lamination at specific desired locations, thereby preventing contamination caused by human intervention. In particular, when employing an AI-assisted automation system, it is possible to utilize the flexibility of the 6-axis robotic arm and perform mixing and lamination (random or patterned) of multi-component bioinks on cell culture plates. Generally, time-based or volume-based (flow control) control methods can be used. However, accuracy is difficult to expect with both of these methods due to the high viscosity of hydrogel bioinks, pressure differences occurring during multi-point lamination, and the viscosity of the hydrogels. However, the feedback control mechanism used in the preferred embodiment of the present invention utilizes an indirect technique that continuously monitors the lamination weight of the hydrogel and converts it into volume, immediately stopping lamination when a set value is reached. Therefore, this lamination control method significantly improves the precision and accuracy of multi-position lamination compared to the two aforementioned methods, whether in a pneumatic or two-screw-based system integrated with a 6-axis robotic arm.

[0020] In addition, according to one embodiment of the present disclosure, by automating operations through a 6-degree-of-freedom robotic arm and a central control unit, the labor time of researchers can be reduced and the input of bio-labor can be minimized. Furthermore, the present invention is applicable to various industrial fields in the regenerative medicine industry, such as bioprinting of organs and tissues, printing of damaged tissues, cell therapy transplantation, 3D cell culture, and drug delivery, thereby having the effect of simultaneously increasing research efficiency and reliability.

[0021] However, the effects of the present invention are not limited to those mentioned above, and other unmentioned effects will be clearly understood by those skilled in the art from the description below.

[0022] FIG. 1 is a schematic diagram of an automated quantitative control dispensing system using interchangeable extrusion heads (robotic arm-based pneumatic and Biowork Pen®) mounted on a 6-degree-of-freedom robotic arm.

[0023] FIG. 2 is a block diagram of an exemplary embodiment for explaining the schematic configuration of the robotic arm bioprinting automation system of the present invention.

[0024] FIG. 3 is a flowchart illustrating the control flow of an embodiment of the robotic arm bioprinting automation system of the present invention.

[0025] FIG. 4 is a schematic diagram illustrating the steps for preparing the configuration of an embodiment of the robotic arm bioprinting automation system of the present invention.

[0026] FIG. 5 is a schematic diagram illustrating the step of selecting a cell culture plate in the configuration of an embodiment of the robotic arm bioprinting automation system of the present invention.

[0027] FIG. 6 is a schematic diagram illustrating an exemplary configuration of a robot arm system equipped with a Biowork Pen for bioink deposition according to the present invention.

[0028] FIG. 7 is a schematic diagram illustrating the configuration of an exemplary embodiment of a robot arm system equipped with a pneumatic syringe of the present invention.

[0029] FIG. 8 is an image showing the configuration of an exemplary embodiment of a 6-well plate injected with the culture medium of the present invention.

[0030] FIG. 9 is an image illustrating an example of the cell culture medium replacement step of the present invention.

[0031] FIG. 10 is an image illustrating an embodiment of a robot arm attachment for the Biowork Pen and pneumatic extrusion system of the present invention.

[0032] FIG. 11 is a circuit diagram of an embodiment when the Biowork Pen of the present invention is connected to a robot arm.

[0033] FIGS. 12a to 12e are circuit diagrams of an embodiment of the pump, timer relay, and valve connection of the present invention.

[0034] FIG. 13a is a circuit diagram of an embodiment of weight measurement and deposition time control using Python code on data collected using the load sensing resistor and Arduino processor of the present invention.

[0035] FIG. 13b is an image showing the output result of an embodiment using Python code on data collected using the load sensing resistor and Arduino processor of the present invention.

[0036] FIG. 13c is a circuit diagram of a weight measurement and deposition time control circuit of an embodiment using Python code on data collected using the load cell, amplifier (HX711), and Arduino processor of the present invention.

[0037] FIG. 14 is a graph showing the results of an example of a hydrogel deposition test of the present invention.

[0038] FIG. 15a is an image showing a robot arm of one embodiment of the load control experiment of the present invention (bioink adhesion / deposition experiment using a Biowork Pen attached to a robot arm) and hollow-shaped deposition (printing) on ​​a 6-well plate using Greensonic (commercial) gel.

[0039] FIG. 15b is an image showing the deposition (printing) result of a 4% alginate hydrogel ionically bonded to calcium ions containing cells in a 6-well plate using a Biowork Pen of one embodiment of the load control experiment of the present invention (a bioink gradual experiment using a Biowork Pen attached to a robot arm).

[0040] FIG. 15c is an image showing the deposition (printing) result of a 4% alginate hydrogel containing cells in a 6-well plate along with cell culture medium using the Biowork Pen of the present invention.

[0041] FIG. 15d is an image showing the day 0 cell viability staining results inside the bioink {1 million / ml osteoblasts (MC3T3) loaded in a 4% alginate hydrogel matrix} deposited in a 6-well plate of one embodiment as a result of evaluating cell viability in the bioink deposited using the robotic arm of the present invention.

[0042] Figure 16 is a diagram showing the results of analyzing the functional group characteristics and crystal structure of hydrogel compositions that include or do not include inorganic particles.

[0043] Figure 17 illustrates the mechanical texture characteristics, repetitive compression characteristics, and viscosity changes according to shear conditions of an alginate-based hydrogel and an oxidized carboxymethylcellulose-chitosan (OCMC-CS) hydrogel.

[0044] Figure 18 is an image showing the results of analyzing the microstructure and elemental distribution of alginate-based hydrogels and oxidized carboxymethylcellulose-chitosan (OCMC-CS)-based hydrogels with or without inorganic particles using scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS).

[0045] Figure 19 shows the distribution characteristics of the dispensing amount when an alginate hydrogel is dispensed based on pressure and time using a pneumatic extrusion head.

[0046] Figure 20 is a figure showing the distribution characteristics of the dispensing amount when a hydrogel composition is dispensed into multiple wells of a multi-well plate by an extrusion method, and the dispensing deviations in the row and column directions relative to the overall average.

[0047] Figure 21 is a diagram showing the distribution of the actual dispensing amount relative to the target dispensing amount when a hydrogel composition is dispensed into a multi-well plate using a pneumatic extrusion system and closed-loop quantitative control.

[0048] FIG. 22 is a diagram illustrating the distribution characteristics of the dispensing amount according to whether feedback control is applied when a hydrogel composition is dispensed into a multi-well plate using a screw-based extrusion head mounted on a robot arm.

[0049] Figure 23 illustrates the distribution characteristics of the dispensing amount when an oxidized carboxymethylcellulose-chitosan (OCMC-CS)-based hydrogel composition is dispensed into a multi-well plate using a pneumatic extrusion system with feedback control and a screw-based extrusion system.

[0050] Figure 24 is a figure illustrating a comparison of cell distribution and viability when multi-component hydrogel bioinks are treated with different mixing methods.

[0051] Figure 25 illustrates the cell viability, cell growth characteristics, and cell distribution status according to the culture period in an in vitro culture test using an alginate-based hydrogel and an oxidized carboxymethylcellulose-chitosan (OCMC-CS)-based hydrogel.

[0052] The present invention will be described in detail below with reference to the embodiments and drawings. These embodiments are presented merely as examples to explain the invention more specifically, and it will be obvious to those skilled in the art that the scope of the invention is not limited by these embodiments.

[0053] Furthermore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention pertains, and in the event of a conflict, the description in this specification including definitions shall prevail.

[0054] To clearly explain the proposed invention in the drawings, parts unrelated to the description have been omitted, and similar parts throughout the specification have been given similar reference numerals. Furthermore, when a part is described as "comprising" a certain component, this means that, unless specifically stated otherwise, it does not exclude other components but may include additional components. Additionally, the term "part" as described in the specification refers to a single unit or block that performs a specific function.

[0055] All technical terms used in this invention, unless otherwise defined, are used in the sense generally understood by those skilled in the art in the relevant field of this invention. Additionally, while preferred methods or samples are described herein, similar or equivalents are also included within the scope of this invention. The contents of all publications cited as references in this specification are incorporated by reference in their entirety into this invention.

[0056]

[0057] Hereinafter, embodiments and examples of the present invention will be described in detail with reference to the attached drawings. However, the present invention may not be limited to these embodiments and examples and drawings.

[0058]

[0059] An automated quantitative control bio-dispensing system according to one embodiment of the present invention (hereinafter “system”) may be composed of an integrated system comprising: (i) a multi-axis robot arm (e.g., a 6-degrees-of-freedom (6 DOF) robot arm), (ii) a dispensing head (extrusion head) module that is replaceably mounted on the end effector of the robot arm, (iii) a real-time weighing unit (including a weight measuring unit) that measures the weight change of the bio-ink in real time during dispensing, (iv) an image acquisition device (camera), and (v) a central control and processing unit (controller).

[0060] The robot arm is configured to control the dispensing position, angle, and movement path in a three-dimensional space and may be configured, for example, with three or more axes (e.g., 3, 4, 5, 6, 7 axes or more). The working range of the robot arm may be set, for example, within a range of 0.01 mm to 2000 mm and may be appropriately changed depending on the work target (plate type, well arrangement, stage size, etc.).

[0061] The end effector of the robot arm may be equipped with a holder or connecting device for mounting a dispensing head (extrusion / suction module), and the holder may be formed of a metal, non-metal, or plastic material. Depending on the purpose of the operation, the dispensing head module may be configured to include at least one of a screw extrusion method, a pneumatic syringe method, or a piston (mechanical) drive method, and multiple types of modules may be interchangeably used as needed.

[0062] The central control and processing unit described above may be configured to integrally control the robot arm's trajectory, dispensing position, dispensing sequence, dispensing pattern, and dispensing conditions (e.g., pressure, time, screw rotation speed, piston drive amount, etc.). The central control and processing unit may be electrically connected to the robot arm and each module (dispensing head, valve, pump, load cell / amplifier, etc.) via general-purpose input / output pins (GPIO), communication ports (e.g., USB, serial, Ethernet), or wireless communication (e.g., Wi-Fi, Bluetooth).

[0063] In one embodiment of the present invention, the system can perform weight-based closed-loop control by using weight information received in real time during dispensing from a sample stage (weight measuring unit), calculating an error with respect to a target dispensing amount (volume or weight), and dynamically correcting the driving conditions of the dispensing head (e.g., pneumatic extrusion time / pressure, screw motor rotation speed / operation time, piston driving amount, etc.) so that the error converges within a preset allowable range.

[0064] Such weight-based closed-loop control can improve dispensing accuracy and repeatability by compensating for rheological variability, such as shear thinning, back pressure changes, and flow rate changes due to residual amount changes, which may occur in hydrogel bioinks with viscoelastic and non-Newtonian fluid properties. In addition, it can control the dispensing operation to automatically stop and move the robot arm to the next position when the dispensing reaches a specific target weight.

[0065] The sample stage may include a support structure on which a cell culture plate or a target container (hereinafter referred to as the “dispensing target”) is placed, and may include a weight measuring unit (e.g., a load cell, a load sensing resistor, a load amplifier, an ADC module (e.g., HX711), a microcontroller, etc.) for measuring the dispensing weight in real time. The measurement value of the weight measuring unit may be continuously monitored by a central control and processing unit.

[0066] The above sample stage may be configured to measure a load in the range of 0 g to 500 g with, for example, a resolution of 1 mg, and the resolution and measurement range may be appropriately changed depending on the application field and the dispensing amount range.

[0067] The above system may include an image acquisition device (e.g., a webcam) for capturing a workspace, and the image acquisition device may be connected to a central control and processing unit via USB, Wi-Fi, Bluetooth, or other communication methods.

[0068] The central control and processing unit can process image data in a (i) rule-based (e.g., marker / ROI-based, predefined coordinate transformation) or (ii) learning-based (e.g., machine learning-based image classification / object recognition) manner to recognize the type of dispensing target (e.g., 6 / 12 / 24 / 48 / 96*?*well plate), well arrangement, dispensing area, reference coordinates, etc. Based on the recognition results, it can generate a dispensing position model for the robot arm and be configured to automatically execute movement and dispensing operations of the robot arm.

[0069] For example, as illustrated in FIGS. 4 and 5, if a region of interest (ROI) is set in the camera image and a dispensing target plate is placed at a reference position within the ROI, the central control and processing unit can identify the type of plate and automatically select and execute a corresponding dispensing program. If necessary, in addition to commercial standard plates, custom plates may be included.

[0070] A dispensing head module according to one embodiment of the present invention can perform the function of dispensing (extruding) or sucking in and removing fluid materials such as hydrogel, bioink, and culture medium. The dispensing head module can be interchangeably mounted on an end effector of a robot arm and may include, for example, at least one of the following methods.

[0071] The screw-based extrusion module may include a drive motor and a screw (single screw, double screw, or composite screw) and may be configured to extrude while mixing high-viscosity or multi-component bioinks. The motor rotation speed (rpm), operating time, and whether to reverse rotation, etc., may be controlled by a central control and processing unit and may be synchronized with the movement of a robot arm.

[0072] FIG. 6 is a schematic diagram illustrating an exemplary configuration of a robot arm system equipped with a Biowork Pen for bioink deposition according to the present invention, and FIG. 7 is a schematic diagram illustrating an exemplary configuration of a robot arm system equipped with a pneumatic syringe according to the present invention.

[0073] As a preferred embodiment of the present invention, experiments were conducted using a digital Bio Work Pen (Biowork Pen®, a registered trademark) improved by the applicant and the inventor of the present application. The robot arm and the improved digital Bio Work Pen transmit and receive control commands and data using a pin-level serial communication protocol. For this control operation, the system is configured to directly interface with a Python-based automation script to execute extrusion commands, and it was confirmed that stable and reproducible signal transmission is possible. In addition, a USB webcam was placed above the deposition field to implement visual verification of pattern fidelity and synchronization with the robot arm's operation.

[0074] The components of this system are briefly described below.

[0075] The robotic arm system for dispensing viscoelastic hydrogel bioink, specifically the 6-DOF robotic arm system, forms the basis of the automated hydrogel dispensing platform. The robotic arm is programmed to perform high-precision, multi-location hydrogel dispensing tasks (e.g., implemented using a Python API). Before each experiment, coordinate mapping of the experimental platform was performed and uploaded to the control software to minimize spatial errors. The control structure enables synchronization of the robotic arm's position information with extrusion commands, allowing for smooth pattern replication even in complex 3D shapes.

[0076] The dual-screw extrusion system (Biowork Pen®) is a device that has been continuously improved through various research processes since its initial development stage. It was first introduced as a screw-based hydrogel extrusion device attached to a 3D bioprinter. The initial model was a manually driven system that applied various pitches to a screw-barrel structure to achieve uniform mixing and precisely dispensed multi-component bioinks on demand while minimizing damage to living cells, thereby demonstrating the potential of the digital Biowork Pen® in localized tissue engineering applications. Subsequent research improved the internal extrusion mechanism, and through a design combining dual-screw and single-screw structures, it evolved into a highly portable mixing and dispensing device that can be used as an attached to a 3D bioprinter or as a portable unit. Furthermore, the Biowork Pen underwent several structural and control changes during the process of integration with a robotic arm.

[0077] -1st Generation: Manual extrusion via potentiometer-based DC voltage regulation and toggle switches—low cost and practical, but had low positional and volumetric precision and required constant human intervention.

[0078] -2nd generation: Addition of Bluetooth and Wi-Fi-based interfaces → Wireless control of screw motor speed and dispensing timing is possible. However, the screw-barrel structure and sterilization / biosave protocols remain the same.

[0079] As described above, the digital biowork pen was improved and integrated with a 6 DOF robotic arm, and a Python-based closed-loop software system was built to control it. When the user sets the screw rotation speed and dispensing speed, the robotic arm is configured to synchronize with spatial coordinates and perform dose-specific dispensing at designated points. This approach is particularly useful for repetitive and large-scale patterning tasks as it enables automated bioink supply while minimizing human intervention. The robot platform is equipped with a servo motor (MG996R) connected to a microcontroller (M5 Stack) to control screw operation. The control signal (PWM signal) is transmitted via a serial interface and synchronized with the movement commands of the robotic arm. The screw rotation speed (rpm) is the factor that has the greatest influence on hydrogel mixing and extrusion, and since it varies depending on the power supply voltage, the rotation speed (rpm) is recorded separately under no-load conditions, alginate hydrogel loading conditions, and commercial gel (Greensonic) loading conditions.

[0080] Next, regarding the pneumatic extrusion and suction-extrusion system, the pneumatic extrusion head consists of an adjustable (0-999 kPa) pneumatic regulating valve, a compressor supplying it, and a digitally controlled solenoid valve. The solenoid valve is interfaced via a relay module and operates using a Python-based command protocol. A syringe-based reservoir containing hydrogel is fixed in a custom-made 3D-printed housing, and the outlet is equipped with replaceable nozzles and needles (20G, 22G). During each dispensing cycle, the robotic arm moves to preset XYZ coordinates, and the solenoid valve operates for a set time or until a feedback-based quantitative target is reached. This integrated structure enables optimal patterning performance by dynamically adjusting extrusion conditions such as pressure, needle gauge, and operating time.

[0081] In addition, a full-scale integrated system for the development of an automated cell culture platform was constructed and validated using a suction / injection conceptual model. This system is designed to automatically perform tasks such as medium exchange, reagent delivery, and sample collection in multi-well plates. To this end, it is equipped with a microcontroller that drives small diaphragm pumps and solenoid valves according to preset volume and time conditions. While the mechanical hardware maintains a modular design, the control logic is precisely tuned to ensure spatial coordination with the robotic arm.

[0082] Meanwhile, to increase the level of automation and minimize user intervention, a machine learning module was developed that can automatically recognize cell culture plate types and dynamically select programs in a bio-dispensing system. This acquires an upper image of the workspace in real time using a webcam fixed above the dispensing stage, and the image is processed by a lightweight convolutional neural network (CNN). The CNN is trained on a dataset labeled for common plate types such as 6-, 12-, 24-, 48-, and 96-well plates.

[0083] When a plate is successfully classified, the ML module automatically invokes a robotic program corresponding to the recognized plate format, which includes predefined dispensing coordinates, dose targets, and extrusion parameters. This intelligent plate recognition and response system shortens experiment preparation time while reducing user errors prone to occurring during manual selection. Furthermore, the image dataset can be expanded to include partially occluded plates and plates with irregular arrangements, and it provides object detection capabilities to enable recognition even in layouts containing multiple plates. Additionally, by introducing optical flow-based tracking, it is possible to verify deposition alignment in real time.

[0084] The motor RPM can be set in a range of, for example, 0 to 1000 RPM and can be appropriately adjusted according to the viscosity of the bioink, the target dispensing amount, and the nozzle (needle) specifications. In addition, motor driving conditions can be corrected in real time to reach the target weight through weight-based closed-loop control.

[0085] The pneumatic module may include a suction compression pump, a controller, a pneumatic valve (e.g., a 3 / 2 or 5 / 2 solenoid valve), a timer relay, tubing, a syringe, and a needle (nozzle). The module may be configured to operate in a pressure range (e.g., 0.01 kPa to 1000 kPa) and a vacuum range (e.g., 1000 mbar to 0.1 mbar), and the valve and pump may be controlled by electrical signals from a central control and processing unit.

[0086] The amount of fluid dispensed (extruded) or sucked may be set to time-based open-loop control, or to closed-loop control in which execution time or pressure conditions are dynamically corrected using feedback from the weighing unit.

[0087] The piston-driven module may include a drive motor, a lead screw, a rail / track, a syringe holder, a syringe, and a needle, and can perform extrusion or suction by moving the piston back and forth by motor drive. The lead screw pitch (e.g., 0.1 mm to 10 mm), motor rpm (e.g., 0.1 to 500 rpm), etc., can be set according to the design and application. In addition, the drive time can be corrected in real time using weight-based feedback.

[0088] The power supply for driving each of the above modules may be provided from a power adapter, AC input, or ports of the robot arm and central control unit, and may be distributed to each driving unit (motor, valve, pump, relay, etc.) via a microcontroller as needed.

[0089] The system of the present invention can process various materials such as living cells, culture media, polymers, gels, nano / microparticles, biomolecules, crosslinking agents, and mixtures thereof. Gels may include, for example, self-bonding gels, pre-crosslinked hydrogels, in situ hydrogels, photocurable (e.g., UV-curing) gels.

[0090] The system may be operated in a sterile, non-sterile, controlled, semi-controlled, or uncontrolled environment and, if necessary, may be placed within an environment chamber where temperature (e.g., -20°C to 100°C), relative humidity (e.g., 0% to 100%), and a specific ratio of gas mixture are controlled. A robotic arm may be configured to perform dispensing and culture medium exchange processes inside the environment chamber.

[0091] According to another embodiment of the present invention, the robot arm end effector may be additionally equipped with one or more ultraviolet (UV) or laser light sources and configured to ensure structural stability by inducing immediate photocrosslinking of the photocurable bioink immediately after extrusion.

[0092] In one embodiment of the present invention, the system can perform an automated process that includes not only the mixing and dispensing processes of bioink / hydrogel, but also the processes of adding, removing, and exchanging liquids (medium / washing solution) during the cell culture process.

[0093] For example, the following processes can be repeated according to a schedule: (i) placing a dispensing target (e.g., cell culture plate) on a sample stage and initializing weight measurement; (ii) recognizing the plate type and well location using camera images; (iii) mounting a selected dispensing head (screw / pneumatic / piston) on a robot arm to dispense bioink at a set position and pattern; (iv) spraying a cleaning solution if necessary, then suctioning and removing it to move to a waste tank; and (v) suctioning fresh culture medium from a storage tank and quantitatively injecting it into each well.

[0094] According to one embodiment, the present invention may include an automated process maintained by a central control and processing unit or a graphics processing unit, which uses a camera to detect a deposition location in a workspace on a sample stage, uses a mixing and deposition system attached to a robotic arm to mix and deposit a multi-component bioink containing living cells at the detected location in a desired pattern and amount, and uses the same robotic arm to add, remove, and replace a desired amount of liquid according to a schedule planned for tissue engineering purposes.

[0095] The central control and processing unit described above can generate a deposition location model by capturing camera images of the workspace above the sample stage and training an image classifier via machine learning using labeled samples, and the generated model controls the movement of the robot arm to execute the entire process or a specific part. Additionally, the generated model can be configured to update other controls to execute a target task.

[0096] The above deposition location can be located within a three-dimensional space of 0.01 mm to 2000 mm on each side.

[0097] The location is specified using the example placed on the sample stage above.

[0098] The above example may be a cell culture plate having 1, 2, 4, 6, 12, 24, 48, 96, 192, or 384 wells.

[0099] The above embodiments may be hollow solid objects having boundary walls defined in tubular, cylindrical, rectangular, or other regular and irregular shapes capable of depositing, providing, and retaining hydrogels, bioinks, or liquids.

[0100] The above irregular shape may be a shape similar to damaged bone, cartilage, skin, muscle tissue and organs, or a combination of two or more areas.

[0101] The process according to one embodiment of the present invention may be the entire process or part of the entire process of mixing, deposition, liquid deposition, liquid extraction, or replacement with a fresh liquid of bioink, hydrogel, or other materials.

[0102] According to one embodiment of the present invention, the mixing of various components and the deposit can be achieved through a suitable extrusion mechanism, such as a screw, pneumatic, mechanical pressure driven, or other system attached to the end of a robot arm.

[0103] The above-mentioned deposition may be patterned in different shapes at different locations or may have a patternless shape.

[0104] According to one embodiment of the present invention, the addition, removal, and / or exchange of liquid at a specific location can be achieved using a suitable suction-compression mechanism, such as a screw, pneumatic, mechanical pressure driven, or other system attached to the end of a robot arm.

[0105] The entire process of the present invention can be carried out in a sterile, non-sterile, controlled, semi-controlled, or uncontrolled environment.

[0106] The robot arm may be composed of 3, 4, 5, 6, 7, or more axes.

[0107] The end of the above-mentioned robot arm is equipped with a suitable holder or connecting device for securing an extrusion or suction device.

[0108] The movement of the above robot arm can be controlled using a central control and processing unit.

[0109] The working range of the robot arm is preferably 0.01 mm to 2000 mm.

[0110] The mixing and co-extrusion system or device for the extrusion deposition of the above liquid, hydrogel, bioink, or similar material may be a specially designed screw extruder composed of a drive motor and related accessories.

[0111] The above drive motor is controlled by a central control and processing unit using general-purpose input / output pins, other pins or ports attached to the robot arm, and can synchronize the working time and rotations per minute of the robot arm.

[0112] The central control and processing device described above can set a real-time value for the motor's working time based on feedback received from the weight change of the sample stage mentioned in claim 1.

[0113] The above device or extruder head can be used to mix multicomponent materials, including culture media, polymers, gels, nano and microparticles, and live cells, and to uniformly feed them to another extrusion system, wherein feeding can be achieved by methods other than screw extrusion (e.g., pneumatic, including piston).

[0114] An extrusion system or device for extrusion deposition of the above liquid, hydrogel, bioink, or similar material may be as follows:

[0115] i. It may include a single, double, or composite screw (screw) of general or special design, consisting of a drive motor and related accessories.

[0116] a) The above drive motor can be controlled by a central control and processing unit using a general-purpose input / output pin and a pin or port for working time and revolutions per minute attached to the robot arm to synchronize the movement of the robot arm.

[0117] b) The above motor rpm is preferably 0 to 1000 rpm.

[0118] c) The central control and processing device can set a real-time value for the motor's working time based on feedback received from the weight of the sample stage mentioned in claim 1.

[0119] ii. It may include a pneumatic syringe-based system composed of a suction-compression pump, a controller, a pneumatic valve, a timer-relay, tubing, a syringe, and an extrusion needle.

[0120] a) The above system may be controlled by a central control and processing unit that uses a general-purpose input / output pin, other pins or ports attached to the robot arm for suction, compression, or hold modes, power, and a working time that synchronizes the movement of the robot arm.

[0121] b) The amount of liquid injected or sucked into a designated location can be controlled by a central control and processing unit according to a set operation or execution time.

[0122] c) The above control can be performed directly according to the pump's power and operating time, valve movement, timer relay function, or a combination thereof.

[0123] d) The central control and processing device can set a real-time value for the operation or execution time based on feedback received from the weight of the sample stage.

[0124] e) The pressure range of the above pump is 0.01 kPa to 1000 kPa, and the vacuum range is 1000 mbar to 0.1 mbar.

[0125] f) The above pneumatic valve is an electrically controlled valve such as a 3 / 2 or 5 / 2 solenoid valve or other similar product suitable for control by an electric trigger generated by a central control and processing unit, and the pressure range is 0.01 kPa to 2000 kPa.

[0126] g) The above timer relay is electrically controlled and suitable for control by an electrical trigger generated by a central control and processing unit, and the time interval can be adjusted from 0.1 seconds to 999 minutes.

[0127] h) Any suitable product available on the market can be used for attaching the above syringe and needle to the pneumatic system.

[0128] iii. It may include a mechanically driven (or piston-driven) system, or a suitable system composed of a drive motor and a screw, a rail or track, a syringe holder, an extrusion syringe and a needle.

[0129] a) The above drive motor and drive screw are controlled by a central control and processing unit using a general-purpose input / output pin, other pins or ports attached to the robot arm, and the pitch of the screw is 0.1 mm to 10 mm, and the motor rpm range is 0.1 to 500 rpm.

[0130] b) The runtime is based on real-time feedback received from the weight of the sample stage mentioned in claim 1 and synchronizes the movement of the robot arm.

[0131] c) The rail or track and syringe holder are suitable for fixing the syringe and needle in a fixed position and for moving the piston attached to the disposable syringe back and forth.

[0132] d) Any suitable syringe and needle available on the market can be used to attach to the pneumatic system.

[0133] The above sample step is connected to a central control and processing unit via a Universal Serial Bus, Wi-Fi, Bluetooth, or other methods to continuously measure the weight added during deposition.

[0134] The sample stage is equipped with a load cell, a load amplifier, a microcontroller, and a suitable device for measuring added weight or load, and the value is continuously monitored by a central control and processing unit. The sample stage can measure weights from 0 to 500g with a precision of 1mg. When a set weight is reached at a specific location, deposition is stopped and the robot arm moves to another location.

[0135] The above camera may be any webcam connected to a central control and processing unit via Universal Serial Bus, Wi-Fi, Bluetooth, or other methods.

[0136] Suitable power supplies for driving the aforementioned accessories are provided via a microcontroller (programmable or non-programmable) and via any DC power supply, including power adapters, direct AC inputs, or ports on the robot arm and the central control and processing unit.

[0137] The process material for the above extrusion may be any living or non-living material, including living cells, gels, nano or microparticles, biomolecules, polymers, crosslinking agents, and mixtures thereof.

[0138] The above gel may be any gel, such as a self-bonding gel, a pre-crosslinked hydrogel, an in situ hydrogel, or a UV-bonding gel.

[0139] The capacity of the device or extrusion head mentioned above may be 0.1 ml to 300 ml.

[0140] The device mentioned above may be enclosed in a chamber having a controlled environment in which the temperature is controlled from -20°C to 100°C and the relative humidity is controlled from 0% to 100% and a specific ratio of gas mixture is supplied for the purposes of cell culture, maintenance, and tissue engineering.

[0141] The manufacturing materials of the robot arm, extrusion head, its holder, attachments, and gripper mentioned above may be metal, non-metal, or plastic.

[0142] One or more light sources of ultraviolet light or lasers may be attached to the end of the robot arm mentioned above to provide illumination and photocrossing of the extruded deposition material. The light source may be powered by the power source mentioned in claim 7.

[0143] The above-mentioned system, robotic arm, and extruder head may be used to perform continuous, semi-continuous, and batch mixing and 3D printing or bioprinting methods of multi-component materials including polymers, gels, nano and microparticles, biomolecules, and living cells.

[0144] The method may include a method of continuously, semi-continuously, and batch mixing and 3D printing or bioprinting multicomponent materials including polymers, gels, nano and microparticles, biomolecules, and living cells using the system, robotic arm, and extruder head mentioned above, and a method of manually or motorized (robotically) applying to a desired area (smooth and flat or uneven and irregular area) including a living or non-living body for reconstruction or regeneration purposes.

[0145] According to one embodiment of the present invention, a method for continuous, semi-continuous, and batch mixing and 3D printing or bioprinting of multi-component materials including polymers, gels, nano and microparticles, biomolecules, and living cells can be used to encapsulate biomolecules or drugs in a polymer or gel matrix using a robotic arm and an extruder head, thereby enabling the encapsulated biomolecules or drugs to be continuously released to a target site.

[0146] According to one embodiment of the present invention, a method for synthesizing polymer micro or nanoparticles loaded with or not loaded with biomolecules or drugs may be included using a system, a robotic arm, and an extruder head for continuous, semi-continuous, and batch mixing of multicomponent materials including polymers, gels, nano and microparticles, biomolecules, and living cells, and for 3D printing or bioprinting.

[0147] According to one embodiment of the present invention, a method for continuous, semi-continuous, and batch mixing and 3D printing or bioprinting of multicomponent materials including polymers, gels, nano and microparticles, biomolecules, and living cells using a system, a robotic arm, and an extruder head may be included for all purposes other than the processes mentioned above.

[0148] FIG. 2 is a block diagram of an exemplary embodiment proposed for automated aliquoting (biogel / bioink separation) of the present invention, and FIG. 2 is a process flowchart for software decision-making of the present invention. The separation and ejection of the biogel and bioink are made possible by a screw biopen having a reverse rotation function.

[0149] The robot arm of the present invention can be controlled according to the steps described below.

[0150] FIG. 4 shows the preparation step of an embodiment of the present invention, in which a cell culture well plate is weighed using robot control system code and placed on a weighing stage, a camera is installed on the cell culture plate and the camera's output is visualized using Python code. The region of interest is indicated by a yellow box in the sub-window at the top left of the overall camera view in FIG. 4, which is identical to the dimensions of the cell culture plate. The cell culture plate must be placed in the yellow box.

[0151] As shown in FIGS. 4 and 5, when a selected culture plate is placed in the yellow box, the cell culture plate of the present invention detects the cell culture plate used and selects an appropriate program through artificial intelligence. This system can identify commercial standard cell culture plates of 6, 12, 24, 48, and 96 wells. If necessary, it may also include custom plates other than commercial ones.

[0152] The biowork pen, pneumatic, or piston-type extrusion system of the present invention is used for depositing (cell-containing) bioink or (bioactive substance-containing) hydrogel depending on the operator's selection. The extrusion system is attached to a 6-axis robotic arm that initiates deposition with a set amount and pattern given by command control. The deposited weight is continuously measured, and deposition continues until the set weight is reached or the structure is completed. An image of the process set with the biowork pen attachment for bioink deposition is shown below, and the components are as shown in FIG. 5.

[0153] The present invention, in the next step, washes the bioink structures deposited in the wells with antibiotics or other sterile solutions, and a robotic arm draws a set amount of sterile solution from a storage tank and sprays it onto the samples in the wells using a pneumatic syringe-based extrusion system. After a certain period of time, the robotic arm draws the wash solution from the cell culture wells and disposes of it in a disposal tank using a pneumatic syringe-based system.

[0154] As a step of adding cell culture medium according to one embodiment of the present invention, the robotic arm of the present invention extracts a certain amount of cell culture medium from a storage tank using a suction process, and then drops it onto a sample in a well using a pneumatic syringe-based extrusion system. The process setup using a syringe attachment is as shown in the image of FIG. 7 (a robotic arm system including a robot equipped with a syringe, a scan, medium, well plate, stage, etc.).

[0155] In a cell culture medium replacement step according to an embodiment of the present invention, after a certain period of time, a robotic arm draws the cell culture medium from the cell culture well and discards it into a disposal tank as regularly programmed using an air pressure syringe-based system. Subsequently, a set amount of fresh cell culture medium is taken from a storage tank and deposited into the sample in the well using an air pressure syringe-based system. This medium replacement step continues until the study is completed as set by the operator. FIG. 9 is an image showing the medium being drawn from the well and deposited into the disposal tank.

[0156] FIG. 10 shows a Biowork Pen attached to a robot arm that can be exchanged with a pen / pneumatic system according to an embodiment of the present invention. FIG. 9 (a) shows an assembly drawing, (b) shows a Biowork Pen attached to a robot arm, and (c) shows a pneumatic extrusion system attached to a robot arm.

[0157] According to one embodiment of the present invention, a webcam is used and connected to computer control via USB Type A 2.0 (Universal Serial Bus), and a sample stage position is fixed using Python code (opencv and numpi).

[0158] According to one embodiment of the present invention, the Biowork Pen drive motor is controlled by Python code through the general-purpose input / output pins (GPIO) of the robot arm via an L293 driver, and the circuit diagram is as shown in FIG. 11.

[0159] Figures 12a through 12e are circuit diagrams illustrating examples of pump, timer relay, and valve connections. Figure 12a is a circuit diagram showing that the compression valve is controlled by a timer relay, and the timer is controlled by Python code through the general-purpose input / output pin (GPIO) of the robot arm. Figure 12b is a circuit diagram showing that the compression valve is controlled by Python code through the general-purpose input / output pin (GPIO) of the robot arm via an L293 driver. Figure 12c shows an example of a circuit diagram and a time cycle set, showing that the compression and suction valves are controlled by a timer relay, and the timer is controlled by Python code through the general-purpose input / output pin (GPIO) of the robot arm. Figure 11d is a circuit diagram showing that the compression and suction valves are controlled by Python code through the general-purpose input / output pin (GPIO) of the robot arm via an L293 driver. Figure 12e is a circuit diagram showing that the compression and suction pumps are controlled by a timer relay, and the timer is controlled by Python code through the general-purpose input / output pin (GPIO) of the robot arm.

[0160] FIG. 13 relates to a pump of the present invention, where FIG. 13a shows a circuit diagram for weight measurement and deposition time control using Python code on data collected using a load sensing resistor and an Arduino processor, and FIG. 13b shows the output result. FIG. 13c shows a circuit diagram for weight measurement and deposition time control using Python code on data collected using a load cell, an amplifier (HX711), and an Arduino processor. In a preferred embodiment of the present invention, weight-based stacking verification was performed using a precision scale product from Solid Works, Inc. of the United States, equipped with a real-time data logging function to analyze the correlation between the extrusion amount and robot commands. The automatic dispensing system utilizes a feedback mechanism and is operated by the precision scale and the robot controller. While the hydrogel is being dispensed, the scale continuously measures the stacked weight in real time and transmits the measured data to a control computer system via a USB interface. At this time, the weight of the applied hydrogel can be precisely converted into volume by providing a known density for each bioink. Therefore, real-time weight measurements are continuously recorded even during automatic dispensing, and the accurate dispensing volume is calculated by dividing these values ​​by the corresponding density. This ensures accurate and reproducible dispensing throughout the experiment. Consequently, this closed-loop feedback guarantees precise dispensing and minimizes variability in dispensing volume.

[0161] Figure 14 is a graph showing the results of a hydrogel deposition test according to an embodiment of the present invention. In the absence of weight control, the pneumatic compression system administration time, pressure, and needle control show minimal variation and can be used without a weight control system. Variation between tests using the same parameters is also not significant. Even with low doses of the Biowork Pen, the variation is within 10% of the average value. Reverse operation of the Biowork Pen negatively affects the variation and was therefore excluded from further experiments. Since the RPM of the Biowork Pen depends on the input voltage (6V - 30 RPM, 12V - 150 RPM under no-load conditions), two different voltages were attempted. However, for higher doses of the Biowork Pen deposition, weight control is required to reduce variation (Note: Greensonic Gel is a trade name for a propylene glycol-based water-soluble hydrogel for ultrasound).

[0162] For the alliquair deposition test according to an exemplary embodiment of the present invention, the conditions for using a pneumatic extrusion syringe (A) and using a Biowork Pen (B) were set as described below and in the table.

[0163] A. When using a pneumatic extrusion syringe, (a) 6-well plate, Greensonic gel, 6 seconds deposition, 110kPa (21G needle), 100kPa (20G needle), (b) 12-well plate, Greensonic gel, 100kPa (20G needle), 6 seconds and 4 seconds deposition, (c) 24-well plate, Greensonic gel, 4 seconds deposition, 70kPa (20G needle), 70kPa (21G needle).

[0164] B. When using a Biowork pen. (d) 6-well plate, Greensonic gel, 14G needle, 6V-8 sec deposition-reverse motion (wr), 12V-8 sec deposition-reverse motion (wr), 12V-7 sec deposition-no reverse motion (nr), 12V-10 sec deposition-no reverse motion (nr), (e) 6-well plate alginate hydrogel, 14G needle, 12V, 8s and 15s deposition, (f) 12-well plate, alginate hydrogel, 14G needle, 12V, 6s deposition.

[0165] Standard Experimental Condition Specific Condition Experiment Mean Standard Deviation Coefficient of Variation Pneumatic Extrusion (6 well, Greensonic gel) 110kPa 21G 6s 10.07 378 0.01 106 14.9 100kPa 20G 6s 10.179 60.01 298.6 20.186 60.007 43.9 30.173 80.004 12.3 Pneumatic Extrusion (12 well, Greensonic gel) 100kPa 20G 6s 10.198 40.01 487.4 20.197 0.01 457.3 100kPa 20G 4s 10.123 80.009 17.3 20.125 10.007 66.0 Pneumatic Extrusion (24 well, Greensonic gel)70kPa 20G 4s10.11730.023920.4 70 kPa 21G 4s10.05890.006110.3 20.05180.006312.2Biopen extrusion (6 well, 14G, Greensonic gel)6V 8s wr10.11550.0212118.3 12V 8s wr10.190.037819.9 12V 7s nr10.0980.0099.1Biopen extrusion (6 well, 14G, alginate hydrogel, 12 V)8s10.16220.01328.1 20.19010.01296.7 30.18590.01056.1 15s10.69140.05648.1 20.76650.07289.4Biopen extrusion (12 well, 14G, alginate hydrogel)6s10.34550.082823.9 20.35340.059116.7 30.37170.02546.8

[0166] Experimental mean, standard deviation, and coefficient of variation

[0167] FIG. 15 shows the results of a bioink adhesion test using a Biowork Pen attached to a robotic arm according to an exemplary embodiment of the present invention. FIG. 15a shows an image representing a patterned deposition according to the present invention, specifically an image showing hollow cylindrical deposition (printing) on ​​a 6-well plate using a robotic arm and Greensonic gel. FIG. 15b shows the results of deposition of a 4% alginate hydrogel containing cells on a 6-well plate using a Biowork Pen. FIG. 15c shows the results of deposition of a 4% alginate hydrogel containing cells encapsulated in a cell culture medium on a 6-well plate using a Biowork Pen. FIG. 15d shows the results of evaluating the viability of cells within the bioink deposited using a robotic arm. This is an image showing the results of day 0 cell viability staining inside bioink (1 million / ml osteoblasts (MC3T3) loaded in a 4% alginate hydrogel matrix) deposited in a 6-well plate.

[0168] In order to implement an automated mixing and deposition process according to an embodiment of the present invention, the material composition and manufacturing method of a hydrogel bioink are described below. However, this is merely an example to aid in understanding the present invention, and the scope of the present invention is not limited thereto.

[0169] The hydrogel bioink used in the automated quantitative control bio-dispensing system according to one embodiment of the present invention may be an alginate-based hydrogel, an oxidized carboxymethylcellulose-chitosan (OCMC-CS)-based hydrogel, or a composite hydrogel comprising inorganic particles therein.

[0170] In one embodiment, the alginate-based hydrogel may be formed by ionic crosslinking by reacting an aqueous solution containing sodium alginate with calcium chloride, and may include calcium phosphate-based particles as inorganic particles. The inorganic particles may be added by spatula mixing, screw-based mixing, pneumatic mixing, or a combination thereof so as to be uniformly dispersed within the hydrogel.

[0171] In another embodiment, the OCMC-CS hydrogel can be prepared by mixing an oxidized carboxymethylcellulose solution and a chitosan solution, and a composite hydrogel can be formed by including inorganic particles in the mixture. The hydrogel bioink can be prepared immediately before use to prevent premature gelation or degradation.

[0172] The combination of hydrogel and inorganic particles used in the present invention can be distinguished by the code names listed in Table 2.

[0173] CompositionCodeAs synthesized 2%Alginate HydrogelALGAs synthesized OCMC / CS hydrogelOCMC-CSAs synthesized OCP (no gelatin encapsulation)OCPAlginate hydrogel + 0.5% w / v OCP particlesALG-0.5OCPAlginate hydrogel + 1.0% w / v OCP particlesALG-1.0OCPOCMC / CS hydrogel + 0.5% w / v OCP particlesOCMC-CS-0.5OCPOCMC / CS hydrogel + 1.0% w / v OCP particlesOCMC-CS-1.0OCP

[0174] Table 2 summarizes the combinations of alginate-based hydrogels, OCMC-CS-based hydrogels, and OCP particles used in the embodiments of the present invention. Each composition is indicated by a code name corresponding to the composition for the purpose of description and comparison throughout the specification. Here, the code names are for convenience of explanation and should not be interpreted as limiting the scope of the present invention.

[0175] An automated quantitative control bio-dispensing system according to one embodiment of the present invention may be composed of an integrated system including a multi-axis robotic arm, a central control and processing unit, a plurality of dispensing heads capable of deposition and suction, a sample stage, a weight measuring unit, and an image recognition device.

[0176] The robot arm is equipped with interchangeable end attachments, and at least one of a screw-based extrusion head, a pneumatic syringe-based dispensing head, or a piston-driven dispensing head may be optionally mounted on the end attachments. According to this configuration, the system can perform processes for mixing, depositing, aspirating, or exchanging bioink, hydrogel, liquid medium, or a mixture thereof.

[0177] The central control and processing unit described above is configured to control the movement path, dispensing position, dispensing sequence, dispensing time, and dispensing amount of the robot arm, and can transmit driving signals of the dispensing head through the robot arm's general-purpose input / output pins, communication ports, or corresponding interfaces. Such control can be performed via wired or wireless communication methods.

[0178] The above system may include an image acquisition device positioned above or adjacent to the sample stage, and the image acquisition device provides an image for recognizing the position, shape, or type of a target object placed on the sample stage. The central control and processing unit may be configured to set a deposition position or correct the movement of a robot arm based on the image data.

[0179] In addition, the sample stage is equipped with a weight measuring device to measure weight changes in real time during or after the dispensing process. The central control and processing unit can perform closed-loop control to reach a set target weight or target volume by continuing, stopping, or adjusting the dispensing operation based on a signal received from the weight measuring device.

[0180] According to this configuration, the automated quantitative control bio-dispensing system of the present invention can provide a repeatable and highly reproducible dispensing process regardless of the type, viscosity, or composition of the dispensing target, and can be applied to cell culture, tissue engineering, bioprinting, or similar applications.

[0181] In one embodiment of the present invention, the robot arm may be composed of a robot arm having multiple degrees of freedom and may be controlled to perform multi-position dispensing of hydrogel bioink.

[0182] The robot arm is controlled by a central control and processing unit and can correct spatial errors by performing coordinate mapping for the sample stage before dispensing. The control structure can be configured to synchronize the position movement of the robot arm with the extrusion command of the dispensing head, so as to pattern and dispense hydrogels for multiple positions or three-dimensional shapes.

[0183] A dispensing head according to one embodiment of the present invention may be a pneumatic extrusion head, and the pneumatic extrusion head may include an air pressure supply unit, a pressure regulating unit, a solenoid valve, a syringe, and a nozzle.

[0184] The solenoid valve described above can be opened or closed by an electrical signal from a central control and processing unit and can be operated according to dispensing time or weight-based feedback. The syringe-based reservoir can be combined with a replaceable needle or nozzle and can perform dispensing or suction at specific coordinates in conjunction with the movement of a robotic arm.

[0185] In addition, a system according to one embodiment of the present invention may be configured to automatically perform the injection, removal, or replacement of a cell culture medium, and this may be implemented through an inhalation-injection mechanism.

[0186] A dispensing head according to one embodiment of the present invention may be a screw-based extrusion device. The screw-based extrusion device may include a single screw, a double screw, or a combination thereof, and may be configured to extrude while mixing a high-viscosity hydrogel or a multi-component bioink.

[0187] The rotation of the screw is controlled by a drive motor, which is electrically connected to a central control and processing unit and can be synchronized with the movement of the robot arm. The screw rotation speed, operating time, and extrusion conditions can be adjusted according to the viscosity of the bioink or the dispensing target.

[0188] An automated quantitative control system according to one embodiment of the present invention may include a weight measuring unit disposed on a sample stage. The weight measuring unit may measure the weight change of the hydrogel in real time during dispensing and transmit the measured value to a central control and processing device.

[0189] The central control and processing unit can be configured to control the operation of the dispensing head based on real-time weight feedback, and to stop dispensing when a set target weight or target volume is reached. Through such a closed-loop control structure, fluctuations in the dispensing amount can be reduced and reproducibility can be ensured.

[0190] A system according to one embodiment of the present invention may include a camera positioned above a sample stage. The camera transmits an image of the workspace to a central control and processing unit, and the image may be processed by an image recognition algorithm.

[0191] Based on the image recognition result, the central control and processing device can identify the type or arrangement of sample containers and automatically select and execute a dispensing program corresponding thereto.

[0192] Hydrogels and composites according to one embodiment of the present invention can be characterized through spectroscopic analysis, crystal structure analysis, evaluation of mechanical properties and cell culture evaluation.

[0193] The functional group characteristics of the hydrogel can be confirmed through infrared spectroscopy, and the crystal structure can be evaluated through X-ray diffraction analysis. In addition, the viscosity, repeated compression characteristics, and texture characteristics of the hydrogel can be used as indicators to determine dispensing suitability and structural stability.

[0194] The cell viability of the hydrogel containing cells can be evaluated after a certain period of culture, and this is to confirm that the automated mixing and dispensing system of the present invention can process the bioink while minimizing cell damage.

[0195] The functional group characteristics and crystal structure of an alginate-based hydrogel, an oxidized carboxymethylcellulose-chitosan-based hydrogel, and a composite containing inorganic particles prepared according to one embodiment of the present invention were analyzed.

[0196] FIG. 16 is a figure showing the results of crystal structure analysis of (a) an alginate hydrogel containing alginate and inorganic particles, (b) a mixed hydrogel containing an oxidized carboxymethylcellulose-chitosan mixture and inorganic particles, and (c) inorganic particles alone and the hydrogel composite.

[0197] Infrared spectroscopic analysis revealed characteristic peaks attributed to carboxylate functional groups in the alginate-based hydrogel, serving as an indicator of the presence of alginate polymers. Additionally, when crosslinking by calcium ions was formed, changes in absorption bands in specific wavenumber regions were observed, suggesting the formation of ionic crosslinked structures. Peaks attributed to bonding within the alginate backbone were also observed.

[0198] In the case of inorganic particles, additional characteristic peaks attributed to phosphate functional groups were observed, indicating that inorganic particles were incorporated within the polymer matrix. In the case of the oxidized carboxymethylcellulose-chitosan-based hydrogel, positional changes or width expansions were observed in the peaks attributed to amide bonds, indicating that interpolymer interactions were formed. These results serve as grounds to confirm that cross-linked structures were formed and inorganic particles were stably incorporated in both hydrogel systems.

[0199] As a result of X-ray diffraction analysis, characteristic diffraction peaks were observed at specific diffraction angles in samples of inorganic particles alone, and these peaks were also confirmed in hydrogel composites. However, in the case of composites, a decrease in the intensity or broadening of the diffraction peaks was observed due to the influence of the polymer matrix, which can be interpreted as a result of the long-range crystal structure being partially relaxed by the polymer matrix. Nevertheless, it was confirmed that the remaining crystal structure is maintained within the hydrogel.

[0200] Repeated compression tests, texture profile analysis, and viscosity analysis were performed on an alginate-based hydrogel and an oxidized carboxymethylcellulose-chitosan-based hydrogel prepared according to one embodiment of the present invention.

[0201] Figure 17 is a figure showing (a) the repeated compression behavior of an alginate gel, (b) the repeated compression behavior of an oxidized carboxymethylcellulose-chitosan gel, (c, d) texture parameters of each gel, (e) a comparison of cohesiveness, and (f) viscosity change characteristics.

[0202] As a result of repeated compression tests, the alginate-based hydrogel exhibited behavior in which compressive strength gradually changed with repeated application of load, while the carboxymethylcellulose-chitosan-based hydrogel showed a tendency to maintain its structure even after repeated compression. This difference can be interpreted as being due to differences in the hydrogel network formation method.

[0203] Texture profile analysis revealed that hardness, tackiness, and chewability varied depending on the hydrogel composition, and changes were observed in each indicator when inorganic particles were included. This suggests that the inclusion of inorganic particles affects the physical properties of the polymer network. Additionally, cohesiveness analysis confirmed differences in internal bonding stability depending on the hydrogel composition.

[0204] As a result of viscosity analysis, both alginate-based hydrogels and oxidized carboxymethylcellulose-chitosan-based hydrogels exhibited viscosity behaviors that changed with variations in shear conditions, and the decrease in viscosity was relatively large in certain compositions. These viscosity characteristics can affect flow behavior during extrusion or dispensing processes and may also be related to structural retention characteristics after dispensing.

[0205] The results of such mechanical and rheological analysis can be used as data to specifically explain the physical properties that can be considered in the hydrogel dispensing and patterning process using the automated dispensing system of the present invention.

[0206] FIG. 18 is a figure showing the results of observing the surface morphology and composition distribution according to the inclusion of inorganic particles and the mixing method of an alginate-based hydrogel and an oxidized carboxymethylcellulose-chitosan (OCMC-CS)-based hydrogel according to an embodiment of the present invention using scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS).

[0207] Specifically, FIGS. 4(a) and 4(d) show the surface morphology and elemental distribution of an alginate hydrogel and an OCMC-CS hydrogel that do not contain inorganic particles, respectively, and FIGS. 4(b) and 4(e) show the results when inorganic particles are included in the same hydrogel and then mixed by mechanical stirring. In addition, FIGS. 4(c) and 4(f) show the results when hydrogels of the same composition are mixed using a screw-based mixing and extrusion device, respectively.

[0208] SEM observation confirmed that the hydrogel composition according to one embodiment of the present invention forms an overall porous structure and that the internal pores form an interconnected network. When inorganic particles were included, a tendency for the micro-roughness and pore structure of the hydrogel surface to change was observed, and these structural characteristics showed differences depending on the mixing method.

[0209] According to the EDS mapping results, in the composition mixed by mechanical stirring, areas of localized concentration of inorganic particles were observed, whereas when a screw-based mixing device was used, elemental signals attributed to inorganic particles were relatively uniformly distributed throughout the hydrogel. This can be interpreted as being due to differences in the shear action applied during the mixing process and the continuous mass transfer method.

[0210] In particular, it was confirmed that when a screw-based mixing and dispensing system combined with a robotic arm is used, the dispersion state of inorganic particles within the hydrogel matrix is ​​maintained more uniformly, which can contribute to securing reproducible properties in subsequent dispensing, patterning, or structure formation processes.

[0211] The results of such morphological analysis show that the hydrogel composition and the method of preparation and mixing according to one embodiment of the present invention can form a stable dispersion structure in terms of microstructure, and these structural characteristics provide a basis suitable for dispensing processes, pattern maintenance, and subsequent biological applications.

[0212] According to one embodiment of the present invention, time-based dispensing characteristics of a viscoelastic hydrogel were evaluated using a pneumatic dispensing head. FIG. 19 illustrates the results of dispensing an alginate gel under different pressure and operating time conditions. Specifically, the pressure conditions were set to 100 kPa, 120 kPa, and 140 kPa, and the dispensing times were set to 4 seconds, 6 seconds, and 8 seconds. Dispensing was performed on a 6-well plate and a 12-well plate, respectively, and needle sizes of 20G and 22G were used.

[0213] When a 20G needle was used in a 6-well plate, the dispensing amount tended to increase according to the set pressure and dispensing time conditions. Under conditions of relatively short dispensing times, the variation in dispensing amount between wells was observed within a limited range, and it was confirmed that the dispersion of the dispensing amount increased along with the increase in dispensing time. This can be interpreted as being due to the fact that, given the flow characteristics of viscoelastic hydrogels, changes in internal pressure may accumulate as the extrusion time increases.

[0214] When using a 22G needle under identical 6-well conditions, the total dispensing volume decreased relatively with the reduction in needle inner diameter. Particularly under low-pressure conditions, it was observed that the dispensing volume was not sufficiently secured despite the set dispensing time; while the dispensing volume increased with increasing pressure, the fluctuation range between repeated dispensing also tended to increase. These results suggest that needle specifications can affect dispensing stability in pneumatic dispensing.

[0215] When a 20G needle was used in a 12-well plate, different dispensing results were observed compared to a 6-well plate, even under the same pressure and time conditions. This may be attributed to changes in dispensing conditions due to differences in well size and the movement path of the robotic arm. Under some pressure conditions, linearity of the dispensing amount tended to be maintained with increasing time, but under long-duration dispensing conditions, it was also confirmed that the variation between wells expanded.

[0216] When a 22G needle was used in a 12-well plate, relatively stable dispensing results were observed under conditions of relatively short dispensing times. On the other hand, under conditions of high pressure or long dispensing times, it was confirmed that droplet phenomena or flow instability occurred during dispensing.

[0217] From these results, it was confirmed that the dispensing characteristics of a time-based dispensing method using a pneumatic head can be significantly affected by the set pressure, dispensing time, needle specifications, and plate structure. In particular, in the case of hydrogels having viscoelastic and non-Newtonian fluid properties, there may be a range where it is difficult to sufficiently suppress fluctuations in the dispensing amount using only a time-based open control method. Therefore, as described in another embodiment of the present invention, more stable dispensing control is possible by combining it with a closed-loop quantitative control method including weight-based feedback.

[0218] According to one embodiment of the present invention, when dispensing a hydrogel onto a plate having a plurality of wells using a pneumatic dispensing head, the movement path of the robot arm can be set differently depending on the number of wells and the arrangement structure of the plate.

[0219] FIG. 20 shows the process of pneumatically dispensing an alginate hydrogel under constant pressure conditions (e.g., approximately 120 kPa) and the same needle size (e.g., 20G),

[0220] (a) 24-well plate,

[0221] (b) 48-well plate, and

[0222] (c) A diagram showing the dispensing results in a 96-well plate.

[0223] For each plate, the average deviation in the row and column directions was plotted together based on the total average dispensing amount.

[0224] In one embodiment, in an array structure with relatively narrow spacing between wells, such as a 24-well plate, changes in the position correction error of the robot arm and the internal pressure of the dispensing head due to the reduction in the travel distance between dispensing points may affect the variation in the dispensing amount. In particular, as dispensing progresses, the extrusion conditions may fluctuate slightly as the remaining amount of hydrogel inside the dispensing head decreases, and as a result, the average dispensing amount by row or column may show a difference within a certain range from the overall average.

[0225] Accordingly, in one embodiment of the present invention, when the number of wells increases, such as with a 48-well plate and a 96-well plate, a predefined robot arm movement path and dispensing sequence can be applied to configure the movement pattern between dispensing points to be uniform. For example, by performing continuous dispensing in a specific row or column direction, or by setting the dispensing sequence in an alternating manner, the influence of changes in internal conditions of the dispensing head can be dispersed.

[0226] As a result of applying this movement strategy, the row-by-row and column-by-column average dispensing amount in each plate can be maintained within an acceptable range relative to the overall average, and it can be confirmed that relatively uniform dispensing is possible even in multi-well plates. The setting and optimization of this robot arm movement path can be selectively changed depending on the number of wells to be dispensed, the arrangement type, the physical properties of the hydrogel, and the dispensing conditions.

[0227] According to one embodiment of the present invention, a pneumatic extrusion head is combined with an automatic quantitative control system using weight-based feedback to control the hydrogel to approach a target dispensing amount.

[0228] Such an automatic quantitative control system can be configured to dynamically adjust the operating time and extrusion conditions of a pneumatic extrusion head using dispensing weight information received in real time from a weight measuring unit placed on a sample stage.

[0229] FIG. 21(a) illustrates an example in which alginate gel is dispensed into a 6-well plate using a pneumatic extrusion head with the above automatic quantitative control system applied, and the target dispensing amount is set to a range of about 100 μL to 300 μL.

[0230] In this embodiment, when an automatic quantitative control system is applied, it was confirmed that the actual amount dispensed into each well can show a value close to the set target amount even in multiple repeated experiments.

[0231] However, it was observed that in the relatively low target dispensing volume range (e.g., about 100 μL to 150 μL), the deviation from the target value may increase depending on environmental conditions, changes in hydrogel viscosity, or pneumatic response characteristics during the dispensing process. This trend implies that, as illustrated in Fig. 21(c), the dispersion of the dispensing volume per well may appear relatively large under low target dispensing volume conditions.

[0232] On the other hand, as the target dispensing amount increases (e.g., about 250 μL to 300 μL), the frequency of under-dispensing or over-dispensing decreases, and the dispensing amounts per well tend to be more concentrated around the target value. This suggests that pneumatic dispensing based on automatic quantitative control can operate stably over a relatively large capacity range.

[0233] FIG. 21(b) illustrates a dispensing example performed on a 12-well plate under the same automatic quantitative control conditions. In the 12-well plate, compared to the 6-well plate, the variation in dispensing amount between repeated experiments may be smaller even under the same target dispensing amount conditions. In particular, reproducibility between experiments may be further improved in the region of relatively large target dispensing amounts.

[0234] As shown in Fig. 21(d), the actual dispensing amount measured for each well of a 12-well plate can form a relatively consistent distribution relative to the target dispensing amount, which shows that dispensing stability can vary depending on the plate shape and well arrangement.

[0235] Through such embodiments, it is confirmed that when a weight-based feedback-controlled automatic metering control system is applied to a pneumatic extrusion head, dispensing accuracy and repeatability can be improved compared to a simple time-based open-loop control method.

[0236] Therefore, the automatic quantitative control-based pneumatic dispensing method of the present invention can be usefully applied to a process of quantitatively dispensing a material having viscoelastic and non-Newtonian properties, such as a hydrogel, into a multi-well plate.

[0237] As previously explained, pneumatic extrusion heads are suitable for dispensing fluids of relatively low viscosity or hydrogels of simple composition; however, in the case of hydrogel compositions with high viscosity or multiple components, instability of the extrusion flow, fluctuations in dispensing volume, and reduced reproducibility may occur. In particular, for materials with high viscoelasticity and heterogeneous internal composition, such as multi-component bioinks designed for tissue engineering applications, pressure-based extrusion alone has limitations in achieving uniform mixing and stable dispensing control.

[0238] Accordingly, in one embodiment of the present invention, a screw extrusion type dispensing head mounted on a robot arm and driven is applied. The screw extrusion head may include a double screw structure and may be driven by a hybrid control method combining analog and digital control. Through this structure, the mixing and extrusion of high-viscosity hydrogels or multi-component bioinks can be performed simultaneously.

[0239] The screw extrusion head described above is based on a structure previously used as a handheld type or a device attached to a 3D bioprinter, but has been improved to enable automatic position control and dispensing by a robotic arm. Accordingly, the spatial coordinate control of the robotic arm and the screw rotation speed control are synchronized, so that hydrogel can be ejected according to a set dispensing amount at a preset position.

[0240] FIG. 22 illustrates an example of hydrogel dispensing results using a robot arm-based screw extrusion head. FIG. 22(a) shows the results of time-based dispensing to multiple well plates under constant screw rotation speed conditions without applying feedback control. In this case, it can be seen that even under the same rotation speed and operating time conditions, a difference in dispensing amount may occur due to the shape of the well to be dispensed or changes in the internal residual amount as dispensing progresses.

[0241] Figure 22(b) shows the results of dispensing at different screw rotation speeds during the same operating time, demonstrating that the dispensing amount changes with changes in rotation speed. However, due to the characteristics of the high-viscosity hydrogel composition, the relationship between rotation speed and dispensing amount may not necessarily be linear.

[0242] Accordingly, in one embodiment of the present invention, quantitative dispensing was performed by applying weight-based feedback control to the screw extrusion head. FIGS. 22(c) and FIGS. 22(d) show the results of dispensing by applying feedback control within a set target dispensing amount range, demonstrating that the position control of the robot arm and the screw rotation control can be adjusted according to real-time weight measurement results. In addition, FIGS. 22(e) and FIGS. 22(f) show the actual amount dispensed in each well, indicating that results approaching the target dispensing amount can be repeatedly obtained through closed-loop control.

[0243] As such, the dispensing method using a robot arm-based screw extrusion head enables relatively stable extrusion flow and dispensing control even for high-viscosity or multi-component hydrogel compositions, and can be applied in parallel with or selectively with a pneumatic extrusion method. The screw extrusion head according to one embodiment of the present invention can be operated under various conditions depending on the characteristics of the material to be dispensed, the target dispensing amount, the well structure, or the patterning shape.

[0244] The automated quantitative control bio-dispensing system according to one embodiment of the present invention can be applied not only to alginate-based hydrogels but also to OCMC-CS-based hydrogel compositions that have high viscosity and are composed of multiple components.

[0245] Accordingly, feedback control-based quantitative dispensing performance was evaluated for OCMC-CS hydrogel and a composition containing OCP particles in OCMC-CS.

[0246] Figure 23 shows the case where the target dispensing amount in a 6-well plate was set to 100 μL, 200 μL, and 300 μL,

[0247] (a) Pneumatic extrusion head and

[0248] (b) Screw-based extrusion head

[0249] The results of dispensing OCMC-CS and OCMC-CS-1.0OCP hydrogels by applying each are shown.

[0250] In addition, (c) and (d) show the actual dispensing amounts measured per well using each dispensing system.

[0251] In an example using a pneumatic extrusion head, it was confirmed that OCMC-CS hydrogel can be dispensed by volume-based feedback control according to a set target dispensing amount.

[0252] Under the 100 μL target condition, the dispensing amount of the composition containing OCP particles may show a tendency to decrease slightly, but under the 200 μL and 300 μL conditions, a dispensing amount similar to the target value can be secured.

[0253] Meanwhile, in the embodiment using a screw-based extrusion head, OCMC-CS and OCMC-CS-1.0OCP hydrogels can be repeatedly dispensed under the same target dispensing conditions, and the deviation of the dispensing amount between wells at each target volume can be maintained within a limited range.

[0254] As the viscosity of the hydrogel increases with the addition of OCP particles, a tendency for the average dispensing amount to decrease slightly may be observed, but this can be interpreted as being due to an increase in extrusion resistance.

[0255] As shown in FIGS. 23(c) and FIGS. 23(d), when a feedback control-based dispensing system is applied, under medium and high dose conditions of 200 μL and 300 μL, the dispensing amount tends to be concentrated near the target value, while under low dose conditions such as 100 μL, the dispersion may increase relatively.

[0256] This is because it can be affected by the viscoelastic properties of the hydrogel and the resolution of the metering device when dispensing low volumes.

[0257] From the above results, it can be confirmed that an automated quantitative control system including a pneumatic extrusion head and a screw-based extrusion head according to one embodiment of the present invention can perform quantitative dispensing close to the target dispensing amount even for bioinks with high viscosity and complex composition, such as OCMC-CS-based hydrogels.

[0258] In addition, it can be seen that the feedback control function does not act as an intrinsic impeding factor to dispensing precision regardless of the presence of OCP particles, and is applicable under various well structures and target capacity conditions.

[0259] In the case of hydrogel bioinks containing multiple components, the mixing state of the components can affect dispensing stability, dispersion uniformity, and reproducibility in subsequent processes. Accordingly, in one embodiment of the present invention, the mixing state according to different mixing methods was compared for a composition containing inorganic particles in an OCMC-CS-based hydrogel.

[0260] Specifically, a composition containing 0.5 wt% or 1.0 wt% of OCP particles in an OCMC-CS hydrogel was prepared, and mixed by (i) a manual mixing method and (ii) a screw-based mixing method using a digital Biowork Pen® mounted on a robotic arm, respectively. The digital Biowork Pen® is configured to apply shear force while continuously conveying the composition by a screw rotation method.

[0261] The distribution of cells contained in the mixed bioink composition was observed through viability staining (see Fig. 24). In the case of manual mixing, a tendency was observed for cells to be locally concentrated or unevenly distributed in some areas. On the other hand, in the mixing method using the digital Biowork Pen®, a pattern of relatively uniform distribution of cells throughout the composition was confirmed.

[0262] In particular, observations made immediately after mixing (Day 0) confirmed that the cells were relatively evenly dispersed within the hydrogel matrix in the composition mixed using the digital Biowork Pen®. This trend was also observed in the OCMC-CS-1.0OCP composition.

[0263] This can be interpreted as being due to the fact that, as continuous shear force is applied to the composition during the screw-based mixing process, the aggregation or sedimentation of inorganic particles is mitigated and the spatial distribution of constituent components within the composition is stabilized. Furthermore, this mixing method can provide a relatively constant mixing state regardless of the viscosity or component composition of the composition.

[0264] Accordingly, the mixing method using the robot arm-based digital Biowork Pen® according to one embodiment of the present invention is applicable to the mixing process of a hydrogel bioink containing multiple components and can contribute to providing a uniform composition required in subsequent dispensing, patterning, or culture processes.

[0265] According to one embodiment of the present invention, an in vitro cell culture evaluation was performed to confirm the biocompatibility of the alginate and OCMC-CS-based hydrogel bioink prepared using the automated mixing and dispensing system.

[0266] FIG. 25 illustrates the results showing the survival and proliferation behavior of cells encapsulated within a hydrogel mixed and dispensed using a robotic arm-based digital Biowork Pen®. Specifically, FIGS. 25(a) to 25(c) show the evaluation results performed on an alginate hydrogel, and FIGS. 25(d) to 25(f) show the evaluation results performed on an OCMC-CS hydrogel.

[0267] In the case of the alginate hydrogel, MC3T3-E1 osteoblasts were encapsulated in a composition containing calcium phosphate-based particles (OCP) at various concentrations and cultured. As shown in Fig. 25(a), according to the MTT analysis results, cell viability during the culture period showed a tendency to increase over time, and as shown in Fig. 25(b), the daily cell growth rate was also maintained stably. Through the Live / Dead staining image in Fig. 25(c), it can be confirmed that the cells are uniformly distributed within the hydrogel up to day 7 of culture.

[0268] Cell encapsulation and culture were performed on the OCMC-CS hydrogel under the same conditions. As shown in Figures 25(d) and 25(e), the OCMC-CS composition containing OCP exhibited sustained cell viability and growth rates during the culture period, and in particular, an increased cell proliferation trend compared to the initial stage of culture was observed in certain compositions. Figure 25(f) shows the Live / Dead staining results at culture days 0, 3, and 7, demonstrating that the cells maintained a relatively uniform distribution in the hydrogel mixed using the robotic arm-based Biowork Pen®.

[0269] In addition, for the same hydrogel composition, a manual mixing method and a robotic arm-based Biowork Pen® mixing method were compared. As a result, in the mixing method using the Biowork Pen®, a tendency for cells to be more uniformly dispersed within the hydrogel matrix was observed, and a stable distribution state was maintained during the culture period.

[0270] As such, it was confirmed that the alginate and OCMC-CS hydrogel bioinks prepared using the automated mixing and dispensing system according to one embodiment of the present invention provide an environment suitable for cell encapsulation and culture, which supports the applicability of the system to tissue engineering, bioprinting, and cell-based applications.

[0271] An automated quantitative control bio-dispensing system according to one embodiment of the present invention is,

[0272] Multi-axis robotic arm, central control and processing unit, multiple interchangeable dispensing head modules,

[0273] It can be configured as an integrated system including a sample stage, a weight measuring unit, and an image recognition device.

[0274] The above-described robot arm may have six or more degrees of freedom and is configured to precisely control the dispensing position, angle, and movement path within a three-dimensional space. The end effector of the robot arm is equipped with a replaceable holder to accommodate different dispensing methods, allowing for the selective mounting of a screw extrusion type dispensing head or a pneumatic syringe-based dispensing head.

[0275] The above screw extrusion type dispensing head can be configured to mix the multi-component bioink in real time immediately before dispensing or during the dispensing process, thereby preventing premature gelation, component separation, or sedimentation of the hydrogel.

[0276] This real-time mixing structure is particularly advantageous for stably dispensing high-viscosity or multi-component bioinks and provides the effect of minimizing cell damage even when live cells are present.

[0277] The core control strategy of the present invention is not an open control based on division time or pressure,

[0278] It is a closed-loop control method based on real-time weight measurement.

[0279] The central control and processing unit receives real-time weight data during dispensing from a weighing unit integrated into the sample stage, calculates the error from the target dispensing amount, and dynamically corrects the extrusion speed, pressure, or operating time so that the error converges within a preset allowable range.

[0280] This weight-based closed-loop control enables high quantitative accuracy and reproducibility even in hydrogel bioinks with viscoelastic and non-Newtonian fluid properties.

[0281] According to one embodiment of the present invention, the central control and processing device can analyze a workspace image obtained from an image recognition device using a machine learning-based image classification or object recognition model.

[0282] The above machine learning model can be trained to recognize the type of cell culture plate, well arrangement, dispensable area, or surrounding components, and is configured to generate a deposition position model for the robotic arm from this to perform an automatic dispensing process.

[0283] Accordingly, the operator can perform an automated dispensing process without plate selection or coordinate input, and minimize errors caused by user intervention.

[0284] In addition, the system of the present invention, in addition to bioink dispensing,

[0285] It may include an inhalation-injection module that performs automatic removal, replacement, and replenishment of cell culture medium.

[0286] The above module includes a syringe, a pump, a solenoid valve, and a control circuit, and is configured to remove used culture medium from each well and automatically inject a set amount of fresh culture medium under the control of a central control unit.

[0287] According to another embodiment of the present invention, an ultraviolet or laser light source may be additionally mounted on the end effector of the robot arm, and this may be configured to induce immediate photocrosslinking of the photocurable bioink immediately after extrusion to ensure structural stability.

[0288]

[0289] In this specification, only a few examples among the various embodiments performed by the inventors are described; however, the technical concept of the present invention is not limited or restricted thereto, and it is obvious that it can be modified and implemented in various ways by those skilled in the art.

Claims

In an automated system for multi-precision dispensing and cell maintenance of viscoelastic hydrogel bioinks including a 6-degree-of-freedom (6 DOF) robotic arm, (a) An extrusion head module replaceably mounted on the end effector of the above 6-degree-of-freedom robot arm, configured to discharge hydrogel bioink in at least one of a screw extrusion method, a pneumatic method, and a piston drive method; (b) a real-time metering unit that measures the weight change of the hydrogel bioink discharged by the extrusion head module in real time; (c) A closed-loop control unit that dynamically corrects and controls the discharge amount of the extrusion head module based on weight information received from the real-time weighing unit to ensure that the hydrogel bioink reaches a set target dispensing amount; and (d) A central control unit that controls the movement of the above 6-degree-of-freedom robot arm and manages the above closed-loop control unit; An automation system characterized by being composed of including In Article 1, A system characterized by the above-described closed-loop control unit performing weight-based feedback control to correct rheological variability due to viscoelasticity, shear thinning, and back pressure changes of the hydrogel bioink. In Article 1, The system further comprises a camera module for image acquisition, and the central control unit recognizes the position or shape of a dispensing target using image information acquired from the camera module, and generates a dispensing position model of the 6-degree-of-freedom robot arm based thereon, wherein the position recognition and the generation of the dispensing position model are performed by a learning-based control method, a rule-based control method, or a combination thereof. A system according to claim 1, characterized in that the end effector of the 6-degree-of-freedom robotic arm is replaceably equipped with a cell maintenance attachment for suctioning and removing used cell medium and quantitatively injecting fresh medium. A system according to claim 1, characterized in that an ultraviolet (UV) or laser light source for performing photocrosslinking on the extruded hydrogel bioink is additionally attached to the end effector of the 6-degree-of-freedom robot arm. A system according to claim 1, wherein the system is housed within an environment chamber in which temperature, humidity, and mixed gas supply are controlled, and the 6-degree-of-freedom robot arm operates within the environment chamber. A method for multi-precision dispensing of viscoelastic hydrogel bioink using a 6-degrees-of-freedom (6 DOF) robotic arm and a real-time metering unit, (a) A step of setting a target dispensing amount and converting it into a target weight; (b) a step of dispensing the hydrogel bioink to a target location using an extrusion head module mounted on the 6-degrees-of-freedom robot arm; (c) A step of obtaining real-time weight information of the weight of the hydrogel bioink discharged through the real-time weighing unit; (d) a step of dynamically feedback controlling the discharge amount of the extrusion head module until the hydrogel bioink reaches the target weight based on the real-time weight information; and (e) After reaching the target weight, move the 6-degree-of-freedom robotic arm to the next dispensing position or perform a cell medium exchange operation using a cell maintenance attachment; A multi-precision dispensing and automation method for hydrogel bioink characterized by including

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