Process method and device for constructing multi-scale controllable three-dimensional structure

Abstract

The invention discloses a multi-scale controllable three-dimensional tissue construction process method and device. The device comprises a gel small body preparation unit, a three-dimensional mobile platform printing unit and a comprehensive integrated control end. The small gel body preparation unit comprises a controllable power source, a gel storage unit and a hydrogel extrusion element; the controllable power source is used for providing power required by gel extrusion and controlling the extrusion amount and the extrusion speed of hydrogel; the gel storage unit is used for storing a hydrogel matrix; the hydrogel extrusion element is used for extruding the hydrogel matrix to form small gel bodies; the comprehensive integrated control end is used for integrally controlling the output of the controllable power source, the movement of the gel storage unit, the movement of the hydrogel extrusion element and the movement of the three-dimensional mobile platform printing unit, and the accuracy and coordination of the whole printing process are ensured through global integrated control. The flexible preparation of the gel small body with controllable size, variable shape and adjustable material can be realized, and multi-cell, multi-material and multi-scale in-vitro tissue printing construction can be carried out.

Description

Technical Field

[0001] The present invention belongs to the technical field of hydrogel body preparation and biological tissue printing, and specifically relates to a process method and device for multi-scale controllable three-dimensional tissue construction. Background Art

[0002] Currently, the field of tissue engineering and regenerative medicine is developing rapidly, aiming to replace or repair damaged tissues due to disease, injury or other conditions by constructing functional biological tissues. Advances in this field have provided new hope for the treatment of a variety of diseases such as heart disease, diabetes, and joint degeneration. In tissue engineering, hydrogel units play a vital role as a three-dimensional cell culture system. They mimic the natural extracellular matrix, provide cells with necessary support and bioactive signals, promote cell attachment, proliferation, migration and differentiation, and can also regulate cell behavior through their physical and chemical properties. Hydrogel materials in tissue engineering are usually composed of natural or synthetic biocompatible polymers, such as hyaluronic acid, collagen, fibrin, polyethylene glycol (PEG), etc. These hydrogels can be cross-linked by physical or chemical methods to form a stable three-dimensional network structure to maintain the shape and structure of the cell unit. In addition, in this field, bioprinting technology uses biomaterials, cells and other biomolecules to create three-dimensional structures with biological activity. This technology has broad application potential in the fields of medicine, biological research and tissue engineering. Hydrogel bioprinting technology combines the precision of bioprinting with the biocompatibility of hydrogels, and can also control cell distribution and build three-dimensional structures, so it has attracted much attention. With the cross-integration of materials science, biotechnology and engineering technology, hydrogel bioprinting technology is expected to achieve the manufacture of more complex and functional artificial tissues and organs.

[0003] Although hydrogel bioprinting technology has shown great potential in simulating natural tissue structures, it still has many limitations in practical applications. In natural tissues, cells usually appear in the form of clustered tissues rather than in a single cell free state, forming a tissue structure with specific functions. Cell clusters in different types of tissues have different cell densities. For example, the cell density of the liver is approximately thousands to tens of thousands of hepatocytes per cubic millimeter; the cell density of blood vessels depends on the type and size of the blood vessels, and the endothelial cell density of capillaries is approximately 50,000 to 100,000 cells per square millimeter. Therefore, in the process of bioprinting simulation and replication of natural tissues, the size and morphology of the cell unit are crucial to the function of the final tissue. Existing technologies, such as droplet printing technology and two-photon polymerization, can achieve precise placement at the single-cell level, but this technology is often too precise and not suitable for cell group construction and is only suitable for small-scale cell patterning, and it is difficult to expand to the construction of large three-dimensional tissues; extrusion printing is a commonly used bioprinting method, which can print long fibers and tubular structures, but this technology may cause shear damage to cells during the printing process. At the same time, the printing speed of extrusion printing of large-scale complex structures is slow and the precision is insufficient, especially when it comes to alternating printing of multiple materials. Secondly, natural tissues are usually composed of many different types of cells, which interact with each other and complete tissue functions together. However, existing bioprinting technologies have difficulties in printing and managing multiple cell types in one body, which affects the improvement of the biomimetic properties of printed tissues and the reproduction of functions. Although studies have explored controlling the size of gel units by changing printing parameters or using specific equipment, these methods usually require complex operations and it is difficult to achieve integrated rapid printing of multiple materials and multiple cell types. These restrictions have affected the widespread application of hydrogel bioprinting technology and hindered its comprehensive development and practical application in the fields of tissue engineering and regenerative medicine.

[0004] Therefore, in order to meet the requirements of tissue engineering for high precision and functionality, it is urgent to develop a new type of hydrogel printing process and device, which should be capable of manufacturing gel bodies containing cell clusters of various cells and hydrogel materials to meet the printing needs of different types of multi-structure complex tissues. Summary of the invention

[0005] In view of the deficiencies in the above-mentioned prior art, the present invention provides a process method and apparatus for multi-scale controllable three-dimensional tissue construction, which realizes the automated printing and manufacturing of gel bodies containing 10-100 cell clusters as the basic units for in vitro model manufacturing. The gel bodies can have different cell densities, different cell types, different biomaterials and different scale morphologies, ultimately realizing multi-cellular, multi-material and multi-scale heterogeneous construction of in vitro tissues.

[0006] The present invention adopts the following technical solutions:

[0007] On the one hand, the present invention provides a multi-scale controllable three-dimensional tissue construction device, which is special in that:

[0008] It includes a gel body preparation unit, a three-dimensional mobile platform printing unit and an integrated control terminal;

[0009] The gel body preparation unit includes a controllable power source, a gel storage unit and a hydrogel extrusion element;

[0010] The controllable power source is used to provide the power required for extruding the gel and to control the extrusion amount and extrusion speed of the hydrogel; the gel storage unit is used to store a variety of hydrogel matrices carrying different types of cells or bioactive factors; the hydrogel extrusion element is used to extrude the hydrogel matrix to form a gel body;

[0011] The integrated control terminal is used to integrally control the output of the controllable power source, the movement of the gel storage unit, the movement of the hydrogel extrusion element and the movement of the three-dimensional mobile platform printing unit, thereby ensuring the accuracy and coordination of the entire printing process through global integrated control.

[0012] Preferably, the gel storage unit comprises a plurality of gel storage containers and a disc-type gel storage mechanism;

[0013] The multiple gel storage containers are used to independently store different types of hydrogels; the multiple gel storage containers are fixed on the disc of the disc-type gel storage mechanism, and are driven by the disc-type gel storage mechanism to perform circular motion and z-direction motion around the central axis.

[0014] Preferably, the gel storage container comprises a liquid inlet and a liquid outlet;

[0015] The gel storage container is equipped with temperature sensing and control elements, stirring rods, vibrators and capacity sensors. The temperature sensing and control elements can be wirelessly controlled to maintain the appropriate temperature environment required for cell survival, maintain cell activity and gel stability; the stirring rods and vibrators are used to stir and vibrate the hydrogel cell suspension; the capacity sensor is used to monitor the amount of gel in the container in real time to ensure precise control of the required amount of material during the printing process.

[0016] Preferably, the liquid outlet of the gel storage container is controlled and switched by a conical adaptive valve composed of a plurality of elastic sealing sheets; the adaptive valve is acted upon by the gravity of the liquid and the cooperation between the elastic sealing sheets to ensure that the liquid outlet can remain sealed; when the disc-type gel storage mechanism moves and the pipe below is aligned and in contact with the liquid outlet, the top of the pipe can push open the elastic sealing sheet to allow liquid to flow out; the flow rate is controlled by the degree of contact between the top of the pipe and the elastic sheet in the z-direction; once the mechanism moves away from the pipe, the elastic sealing sheet undergoes elastic deformation to automatically restore the closed state, thereby closing the liquid outlet.

[0017] Preferably, the hydrogel extrusion element comprises an extrusion cylinder, an extrusion cylinder and a controllably deformable extrusion head;

[0018] One side of the extrusion barrel is connected to the pipeline outlet, and the hydrogel enters the extrusion barrel through the pipeline; the extrusion barrel is located at the upper end of the extrusion barrel and is used to apply pressure to the gel; the controllable deformation extrusion head is connected to the end of the extrusion barrel, and the gel body is extruded and formed through it.

[0019] Preferably, the extrusion barrel has a piston and a piston shaft, and pressure is generated by the movement of the piston to squeeze the hydrogel matrix from the extrusion barrel to the controllable deformation extrusion head; the extrusion barrel also has a pressure controller, including a pressure sensor and a pressure regulating component, which can monitor the pressure changes inside the extrusion barrel in real time and control the pressure based on the hydrogel properties and printing requirements.

[0020] Preferably, the controllable deformable extrusion head includes a flexible inner wall, which is designed to be polygonal and can be flexibly and highly stretched; a plurality of deformation drivers connected to the flexible inner wall can be pulled outward and retracted, so as to achieve precise size and shape; a plurality of movable arms are respectively connected to the deformation drivers, which provide fixation and can drive the deformation drivers to move to assist them in controlling the size and shape of the extrusion head; an extrusion head controller is used to control the movement of the deformation driver and the movable arm to ensure that the extrusion head is deformed according to a predetermined mode; a flexible stop valve is located at the end of the controllable deformable extrusion head, which can deform with the flexible inner wall and is used to cut off the gel liquid flow to finally form a gel body carrying a cell cluster.

[0021] Preferably, the shape of the extruder head can be changed to a circle, ellipse, oval, rounded rectangle, rounded polygon, irregular circle, triangle, rectangle, rhombus, polygon, etc., and its size determines the size of the gel body, which is determined by the size and density of the cells carried by the gel body.

[0022] Preferably, the three-dimensional mobile platform printing unit comprises a printing platform and a three-dimensional moving mechanism; the three-dimensional moving mechanism is used to control the precise positioning and accurate movement of the printing platform in three-dimensional space.

[0023] Preferably, a cross-linking box is provided on the printing platform, including a plurality of chambers loaded with different liquid cross-linking agents, to help the extruded gel bodies to be cross-linked and shaped; the four walls of the cross-linking box are provided with ultraviolet light generating devices with adjustable light direction, such as lasers, laser diodes, etc., for photo-cross-linking of hydrogels; a temperature control layer is provided under the surface layer of the cross-linking box, which is used for thermal cross-linking of hydrogels and can also ensure appropriate temperature during the printing process.

[0024] Preferably, the controllable power source includes but is not limited to various motors, pneumatic pumps or hydraulic pumps, etc., which can generate power outputs of different frequencies, amplitudes and waveforms, including but not limited to sine wave, square wave, triangle wave and other waveform signals.

[0025] Preferably, the integrated control terminal includes a model parser that can read and parse the input three-dimensional tissue model and convert it into printing parameters; a user interface that allows the user to edit and modify some three-dimensional tissue model printing code parameters, such as printing speed, setting pause time, etc., and also has a real-time preview window that displays the three-dimensional tissue model and printing path, allowing the user to visualize the printing process; a control module that generates detailed optimized printing instructions based on the parameters input by the user and the output of the model parser, while accepting information transmitted by the capacity sensor, and ultimately controlling and coordinating the operation of the controllable power source, disc gel storage mechanism, extruder head controller, stop valve, piston shaft, pressure controller and three-dimensional moving mechanism; a data recording and tracking module that records all key data during the printing process, including time, speed, position and material usage.

[0026] On the other hand, the present invention provides a process for constructing a multi-scale controllable three-dimensional tissue. According to the multi-scale controllable three-dimensional tissue construction device, the method comprises the following steps:

[0027] Step 1, design a three-dimensional tissue model, clarify the purpose, expected function and biological requirements of the tissue model, study and confirm the morphological characteristics of the target tissue, including size, shape, structural hierarchy and cell distribution; determine the required cell type, design the distribution position and density of different cells in the tissue model, then determine the bioactive factor components that need to be integrated and their distribution position, and then select a suitable carrier hydrogel material to support cell growth and tissue function. Finally, it is necessary to design the required tissue size and shape and the appropriate gel body size and shape according to the cell morphology and gel properties. The possible shapes of the tissue include but are not limited to spherical, suitable for simulating cell spheres or certain cell aggregates; cylindrical, suitable for simulating vascular tissue; flat, suitable for simulating epidermis or film-like tissue; it can also be some irregular specific shapes to adapt to different tissues such as wavy, hollow tube, wedge, etc. The shape of the gel body can be a sphere, an ellipsoid, an irregular spheroid, a cube, a cuboid or other polygonal body or a polygonal body with rounded corners, etc., according to the shape of the extruder head. The size of the gel body is usually a few microns to hundreds of microns in diameter or side length according to the size and density of the cells carried.

[0028] Step 2, preparing a gel matrix, mixing the selected cells and bioactive factors with a hydrogel material to prepare a biogel matrix with a suitable cell / bioactive factor density and storing it in a gel body preparation unit, specifically comprising:

[0029] According to the needs of constructing tissues, the required cells and bioactive factors are mixed with hydrogels to prepare a gel matrix, and then the hydrogel matrix material is stored in multiple gel storage containers from the liquid inlet. According to the cell type or active factor and hydrogel material, a temperature sensor and a regulating element are set to provide a suitable temperature to ensure cell activity and gel performance. If the hydrogel matrix needs to be stirred or vibrated, it can be loaded into the corresponding storage container and then processed accordingly. Optional cell-carrying hydrogel materials include but are not limited to hyaluronic acid, alginate, fibrin, collagen, gelatin, polyethylene glycol, polylactic acid-glycolic acid copolymer, methacrylated gelatin, carboxymethyl cellulose and sodium polyacrylate; optional cells include but are not limited to stem cells such as embryonic stem cells, mesenchymal stem cells, etc., fibroblasts, endothelial cells, chondrocytes, cardiomyocytes, hepatocytes, neural cells including neurons and glial cells, endothelial cells, muscle cells and adipocytes, etc.; optional bioactive factors include but are not limited to cytokines, growth factors, extracellular matrix, drugs and hormones, etc.

[0030] Step 3, import the model and start printing. According to the designed tissue model, the integrated control terminal is used to convert it into printing instructions, and the printing parameters can be modified and adjusted through it to start the three-dimensional mobile platform printing unit. During the printing process, the cross-linking box of the printing platform is also required to cross-link and shape the extruded gel bodies in real time to form the required three-dimensional shape of the gel bodies. The specific process is as follows:

[0031] The experimenter needs to determine the tissue type and structure to be printed in advance according to the purpose, design the corresponding three-dimensional tissue model step by step, and then import the three-dimensional tissue model file into the integrated control terminal to generate printing parameters and control instructions, including the circulation sequence volume and flow rate of the gel matrix and the corresponding pressure in the extrusion barrel, the size and shape of the deformed extruder head, and the output mode of the controllable power source (such as sine wave, square wave, etc.), etc. Then, confirm that the imported model is correct in the user interface and preview the printing path. Some parameters can be modified or reset by the experimenter through the user interface of the integrated control terminal according to their own needs and experience. Then, according to the hydrogel cross-linking requirements required for the printed tissue, add the corresponding cross-linking agent to the cross-linking box or perform thermal cross-linking and use a UV light generator for UV cross-linking.

[0032] Step 4, constructing three-dimensional tissues, with multi-type hydrogel matrices as the main body, using controllable deformation extrusion equipment to achieve precise control of the size and shape of the gel bodies, and cooperating with mobile devices, printing the gel bodies carrying cell clusters point by point and layer by layer, and directly and controllably constructing multi-cellular, multi-material, and multi-scale three-dimensional tissues from microscopic to macroscopic levels. The specific process is as follows:

[0033] When the integrated control terminal starts printing, according to the requirements of the constructed cell layer, at different times, the corresponding gel matrix selectively reaches the extrusion barrel from the storage container through the adaptive valve and pipeline. The controllable power source provides power according to the preset mode, and controls the movement of the piston in the extrusion barrel through the piston shaft, thereby squeezing out the gel in the extrusion barrel and forming a gel body through the controllable deformation extrusion head. The extruded gel body is finally cross-linked and fixed in the cross-linking box, and then the three-dimensional moving mechanism accurately positions and moves on the X, Y, and Z axes according to the design model, so as to realize the printing of the gel body layer by layer to finally form the required tissue.

[0034] Compared with the prior art, the present invention has at least the following beneficial effects:

[0035] A process method and device for constructing multi-scale controllable three-dimensional tissues. The device proposed in the process scheme can dynamically adjust the output of a controllable power source and the movement of a deformable extruder head according to a preset mode through an integrated control terminal, and adjust the size and shape of the gel body in real time. In conjunction with a stop valve, gel bodies with variable size, shape, number of cells in a cell cluster, and density can be prepared. The multi-storage container design of the device allows for the simultaneous processing of multiple types of hydrogel materials and cells, provides convenience for multi-material printing, supports integrated printing of multiple cell types, and helps to construct more complex and biomimetic tissue structures. The equipment integrates printing and preparation processes into one In this system, multiple steps from preparation to printing are reduced, the entire workflow is simplified, and rapid printing is allowed before cell activity decreases, which helps to maintain cell vitality and function and improve the quality of printed tissues; the three-dimensional mobile platform of the device can work in conjunction with the integrated control terminal to optimize printing speed and printing accuracy, ensuring that the printed tissue structure has a high degree of consistency and accuracy; the addition of the integrated control terminal simplifies the operating process, improves the technical performance of the printing device, and improves the controllability and coordination of the printing process. Overall, the device has a wide range of overall application scenarios and is more adaptable to the development trend of future bioprinting technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 A schematic diagram of a three-dimensional tissue construction device provided by the present invention;

[0037] Figure 2 A schematic diagram of a gel body preparation unit of a three-dimensional tissue construction device provided by the present invention;

[0038] Figure 3 A schematic diagram of a three-dimensional mobile platform printing unit of a three-dimensional tissue construction device provided by the present invention;

[0039] Figure 4 A schematic diagram of the internal structure of a cross-linking box of a three-dimensional tissue construction device provided by the invention;

[0040] Figure 5 A schematic cross-sectional view of a controllable deformation extruder head of a three-dimensional tissue construction device provided by the invention;

[0041] Figure 6 A schematic longitudinal section diagram of the printing effect of the three-dimensional tissue construction device provided by the present invention;

[0042] Figure 7 This is a schematic cross-sectional diagram of the printing effect of the three-dimensional tissue construction device provided by the present invention.

[0043] in:

[0044] 1-gel body preparation unit, 104-controllable power source, 105-disc gel storage mechanism, 106-controllable deformation extrusion head, 107-extrusion head controller, 108-flexible stop valve, 109-gel storage container, 110-extrusion cylinder, 111-extrusion cylinder, 112-liquid inlet, 113-temperature sensing and regulating element, 114-stirring rod, 115-vibrator, 116-capacity sensor, 117-adaptive valve, 118-pipeline, 119-piston, 120-piston shaft, 121-pressure controller, 122-flexible inner wall, 123-deformation driver, 124-moving arm;

[0045] 2-3D mobile platform printing unit, 201-printing platform, 202-3D mobile mechanism, 203-crosslinking box, 204-ultraviolet light generating device;

[0046] 3-integrated control end, 301-endothelial cell-loaded gel body, 302-pericyte-loaded gel body, 303-astroglial cell-loaded gel body, 304-neuron-loaded gel body, 305-sacrificial cavity gel body, 306-filling gap gel, 307-support gel, 308-cell cluster. DETAILED DESCRIPTION

[0047] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0048] The accompanying drawings show various structural schematic diagrams of the embodiments disclosed in the present invention. These figures are not drawn according to the model design scale, and some details are magnified and some details may be omitted for the purpose of clear expression. The shapes of various regions and layers shown in the figures and the relative sizes and positional relationships therebetween are only exemplary, and may deviate in practice due to manufacturing tolerances or technical limitations, and those skilled in the art may design regions / layers with different shapes, sizes, and relative positions according to actual needs.

[0049] See also Figure 1 and Figure 2 The present invention proposes a multi-scale controllable three-dimensional tissue construction device, including a gel body preparation unit 1, a three-dimensional mobile platform printing unit 2 and an integrated control terminal 3. The gel body preparation unit 1 includes a controllable power source 104, a gel storage unit and a hydrogel extrusion element; the controllable power source 104 is used to provide the power required for extruding the gel and to control the extrusion amount and extrusion speed of the hydrogel; the gel storage unit is used to store a variety of hydrogel matrices carrying different types of cells or bioactive factors; the hydrogel extrusion element is used to extrude the hydrogel matrix to form a gel body; the integrated control terminal 3 is used to control the output of the controllable power source 104, the movement of the gel storage unit, the movement of the hydrogel extrusion element and the movement of the three-dimensional mobile platform printing unit 2, and ensure the accuracy and coordination of the entire printing process through global integrated control.

[0050] The gel bodies can be loaded with various types of active cells, and can also be loaded with drugs and biologically active molecules such as growth factors and exosomes.

[0051] Specifically, see Figure 2 The gel storage unit includes a plurality of gel storage containers 109 and a disc-type gel storage mechanism 105; the plurality of gel storage containers 109 are used to independently store hydrogels of various hydrogel matrices of different cells or bioactive factors; the plurality of gel storage containers 109 are fixed on the disc of the disc-type gel storage mechanism 105, and are driven by the disc-type gel storage mechanism 105 to perform circular motion and z-axis axial motion around the central axis.

[0052] The various hydrogel matrices carrying different types of cells, optional cell-carrying hydrogel materials include but are not limited to hyaluronic acid, alginate, fibrin, collagen, gelatin, polyethylene glycol, polylactic acid-glycolic acid copolymer, methacrylated gelatin, carboxymethyl cellulose and sodium polyacrylate; optional cells include but are not limited to stem cells such as embryonic stem cells, mesenchymal stem cells, fibroblasts, endothelial cells, chondrocytes, cardiomyocytes, hepatocytes, neural cells including neurons and glial cells, endothelial cells, muscle cells and adipocytes, etc.

[0053] Specifically, see Figure 2 The gel storage container 109 includes a liquid inlet 112 and a liquid outlet; a wirelessly controllable temperature sensing and regulating element 113, a stirring rod 114, a vibrator 115 and a capacity sensor 116 are arranged inside the gel storage container 109; the temperature sensing and regulating element 113 can be wirelessly controlled to maintain a suitable temperature environment required for cell survival, maintain cell activity and gel stability; the stirring rod 114 and the vibrator 115 are used to stir and vibrate the hydrogel cell suspension; the capacity sensor 116 is used to monitor the amount of gel in the container in real time to ensure accurate control of the required amount of material during the printing process.

[0054] Specifically, see Figure 2 The liquid outlet of the gel storage container 109 is controlled by a conical adaptive valve 117 composed of multiple elastic sealing sheets; the adaptive valve 117 is under the combined effect of the liquid gravity and the elastic sealing sheets to ensure that the liquid outlet can remain sealed. When the disc-type gel storage mechanism 105 moves and the pipe 118 below is aligned with the liquid outlet, the top of the pipe 118 can push open the elastic sealing sheet to allow the liquid to flow out. The flow rate is controlled by the contact degree between the top of the pipe 118 and the elastic sheet in the z direction. Once the mechanism moves away from the pipe 118, the elastic sealing sheet undergoes elastic deformation to automatically restore the closed state and close the liquid outlet.

[0055] Specifically, see Figure 2 The hydrogel extrusion element includes an extrusion tube 110, an extrusion tube 111 and a controllable deformation extrusion head 106; one side of the extrusion tube 110 is connected to the outlet of the pipeline 118, and the hydrogel enters the extrusion tube 110 through the pipeline 118; the extrusion tube 111 is located at the upper end of the extrusion tube 110, and is used to apply pressure to the gel; the controllable deformation extrusion head 106 is connected to the end of the extrusion tube 110, and the gel bodies are extruded and formed through it.

[0056] Specifically, see Figure 2 The extrusion barrel 111 includes a piston 119 and a piston shaft 120. The movement of the piston 119 generates pressure to squeeze the hydrogel matrix from the extrusion barrel to the controllable deformation extrusion head 106. The extrusion barrel also includes a pressure controller 121, which includes a pressure sensor and a pressure regulating component, and can monitor the pressure changes inside the extrusion barrel 111 in real time and control the pressure based on the hydrogel properties and printing requirements.

[0057] Specifically, see Figure 5The controllable deformable extruder head 106 includes a flexible inner wall 122, which is designed to be a regular octagon and can be stretched and deformed flexibly and highly; eight deformation drivers 123 are connected to the flexible inner wall 122 to pull outward and shrink inward, so as to achieve precise size and shape; eight moving arms 124 are respectively connected to a deformation driver 123, which provides fixation and can drive the deformation driver 123 to move and assist it in controlling the size and shape of the extruder head; the extruder head controller 107 is used to control the movement of the deformation driver 123 and the moving arm 124 to ensure that the extruder head is deformed according to a predetermined mode; the flexible stop valve 108 is located at the end of the controllable deformable extruder head 106, which can deform with the flexible inner wall 122 and is used to cut off the gel liquid flow to finally form the gel body. The above components work together to coordinate so that the extruded gel body can have a variety of sizes and shapes. The deformation driver 123 includes a telescopic rod, which can be pneumatic, electric, hydraulic or shape memory alloy driven.

[0058] As a preferred embodiment of the present invention, the shape of the controllable deformable extrusion head 106 can be changed into a circle, an ellipse, an oval, a rounded rectangle, a rounded polygon, an irregular circle, a triangle, a rectangle, a rhombus, a polygon, etc., and its size determines the size of the gel body, which is determined by the size and density of the cells carried by the gel body.

[0059] Specifically, see Figure 3 The three-dimensional mobile platform printing unit 2 includes a printing platform 201 and a three-dimensional moving mechanism 202. The printing platform 201 is placed on the three-dimensional moving mechanism 202 and moves accurately in three-dimensional space along with the X-axis, Y-axis and Z-axis moving mechanisms.

[0060] Specifically, see Figure 3 and Figure 4 The printing platform 201 is provided with a cross-linking box 203, which is made of transparent material to facilitate the operator to observe the amount and state of the internal liquid cross-linking agent. The cross-linking box 203 includes multiple chambers loaded with different liquid cross-linking agents to help the extruded gel bodies to be cross-linked and shaped; the four walls of the cross-linking box 203 are provided with ultraviolet light generating devices 204 with adjustable light directions, such as lasers, laser diodes, etc., for photo-cross-linking of hydrogels; a temperature control layer is provided under the surface layer of the cross-linking box 203, which is used for thermal cross-linking of hydrogels and can also ensure that the temperature is suitable during the printing process.

[0061] Preferably, the controllable power source 104 includes but is not limited to various motors, pneumatic pumps or hydraulic pumps, etc., which can generate power outputs of different frequencies, amplitudes and waveforms, including but not limited to sine wave, square wave, triangle wave and other waveform signals.

[0062] Specifically, see Figure 2The integrated control terminal 3 is connected to a controllable power source 104, a disc gel storage mechanism 105, an extruder head controller 107, a flexible stop valve 108, a capacity sensor 116, a piston shaft 120, a pressure controller 121 and a three-dimensional moving mechanism 202 through wires, and can integrally control the selective circulation, flow rate, volume and extrusion preparation of the hydrogel and the tissue printing process of the gel bodies.

[0063] The integrated control terminal 3 includes a model parser that can read and parse the input three-dimensional tissue model and convert it into printing parameters; a user interface that allows the user to edit and modify some three-dimensional tissue model printing code parameters, such as printing speed, setting pause time, etc., and also has a real-time preview window that displays the three-dimensional tissue model and printing path, allowing the user to visualize the printing process; a control module that generates detailed optimized printing instructions based on the parameters input by the user and the output of the model parser, and at the same time receives information transmitted by the capacity sensor 116, and ultimately controls and coordinates the operation of the controllable power source 104, the disc gel storage mechanism 105, the extruder head controller 107, the stop valve 108, the piston shaft 120, the pressure controller 121 and the three-dimensional moving mechanism 202; a data recording and tracking module that records all key data during the printing process, including time, speed, position and material usage.

[0064] The three-dimensional tissue model needs to be designed accordingly. First, the purpose and expected function of the tissue model must be clarified, such as repairing specific tissue damage or simulating disease states. Then, according to the purpose and functional requirements, the morphological characteristics of the target tissue, including size, shape, structural hierarchy and cell distribution, are studied. Then, the required cell types are determined, and the distribution position and density of different cells in the tissue model are designed. Then, the bioactive factor components that need to be integrated and their distribution positions are determined. Then, suitable carrier hydrogel materials are selected to support cell growth and tissue function. Finally, with multi-type hydrogel matrices as the main body, heterogeneous multi-cellular, multi-material, and multi-scale in vitro tissue models from micro to macro are constructed layer by layer, trying to simulate natural tissue as closely as possible to ensure the reproduction of tissue functions and ensure that the model design meets the requirements of biology and engineering.

[0065] The heterogeneous multi-cellular, multi-material, multi-scale in vitro tissue can be constructed, including but not limited to skin tissue, cartilage tissue, muscle tissue, heart tissue, vascular tissue, nerve tissue, liver tissue, corneal tissue and adipose tissue, etc., according to the hydrogel material and cell selection. It can also be used for comprehensive printing to construct tumor models and organ chips, etc.

[0066] The present process method and device are specifically applied below by selecting vascularized neural tissue as the construction target.

[0067] A process for constructing a multi-scale controllable three-dimensional tissue, based on the above-mentioned multi-scale controllable three-dimensional tissue construction device, constructing vascularized neural tissue includes the following steps:

[0068] Step 1: Design a three-dimensional tissue model.

[0069] Determine the purpose and intended function of neural tissue models: Construct vascularized neural tissue as a drug testing model.

[0070] Study the morphological characteristics of neural tissue: including the distribution of neurons, glial cells (astrocytes, oligodendrocytes, microglia) and vascular cells (endothelial cells, pericytes).

[0071] Determine the desired cell types and design the distribution and density of different cells in the neural tissue model:

[0072] The tissue centered on the tertiary structure of capillaries is constructed, and the core is made of sacrificial hydrogel material. The outer layer is composed of endothelial cells tightly connected to form the inner wall of the blood vessel. The endothelial cells are flat, followed by pericytes surrounding the outer side of the capillary wall, which covers about 30% of the capillary surface. The outermost layer is a single layer of astrocytes, whose end feet cover about 99% of the blood vessel surface. The capillaries in the brain tissue are surrounded by neurons and a small number of microglia and oligodendrocytes. A single capillary in the brain can supply neurons and glial cells within a few hundred microns in the surrounding area, and the specific coverage range will vary depending on the brain area.

[0073] Select appropriate carrier hydrogel materials: Pluronic F-127 (PF-127) is selected as the core sacrificial material, sodium alginate that is rapidly cross-linked by ionic cross-linking is selected for the rest of the cell-carrying gels, and methacryloyl gelatin that is rapidly cured by visible light is selected to fill the gaps.

[0074] Gel body size and shape design and tissue size: The shape of the endothelial cell and pericyte gel bodies is set as ellipsoids considering the number of cell layers and cell morphology, and the shape of the astrocyte and neuron gel bodies is set as spheres considering the number of cell layers and cell morphology. The endothelial cell gel body has a short axis of 100um, a long axis and a middle axis of 200um and 150um; the pericyte gel body has a short axis of 120um, a long axis and a middle axis of 250m and 200um; the diameter of the astrocyte gel body is 350um; the diameter of the neuron gel body is 450um; the core sacrificial gel body adopts a cylindrical shape, with a primary diameter of 250um and a length of 200um, a secondary diameter of 180um and a length of 150um, and a tertiary diameter of 140um and a length of 100um. The tissue size is set according to the vascular path and the size of the gel body; thickness 2.5mm, length 3mm, width 3mm.

[0075] Step 2: Prepare the gel matrix. The cell density is selected according to the actual situation of the tissue: 5×10 endothelial cells 6 / mL; pericytes: 5×10 4 / mL; astrocytes: 2.5×10 5 / mL; neurons 5×10 3 / mL. After the density is determined, endothelial cells, pericytes, astrocytes and neurons are loaded into the sodium alginate solution, and stored in different gel body preparation units 1 together with methacryloyl gelatin and Pluronic F-127 solutions. After the hydrogel matrix material is stored in the gel storage container 109 from the liquid inlet 112, the storage device where the cell-carrying gel is located is set to a temperature of about 38.5°C, which is the temperature of human brain tissue, and can be vibrated to allow the cells to disperse appropriately. The storage device where the Pluronic F-127 solution is located is set to a temperature of about 15°C.

[0076] Step 3, import the vascularized neural tissue model and start printing. According to the designed tissue model, the integrated control terminal 3 is converted into a printing instruction, including the circulation sequence volume and flow rate of the gel matrix and the corresponding pressure in the extrusion cylinder 111, the size and shape of the deformable extruder 106, and the output mode of the controllable power source 104 (such as sine wave, square wave, etc.). Then, the imported model is confirmed to be correct in the user interface, and the printing path is previewed: the vascularized neural tissue is constructed in a segmented sequence from the center to the outside. In a small segment, the sacrificial tube core is first, followed by the endothelial cell part, the pericyte part, the astrocyte part, and finally the neuron part. Then, calcium chloride solution is added to the chamber of the crosslinking box 203 for crosslinking of the sodium alginate gel, and the temperature of the temperature control layer is set to about 38.5°C. The Pluronic F-127 gel is crosslinked while ensuring cell activity. The ultraviolet light generator 204 is turned on during printing to crosslink the methacryloyl gelatin gel.

[0077] Step 4, constructing vascularized three-dimensional neural tissue, controlling the integrated control terminal 3 to start printing, with the above-mentioned hydrogel matrix as the main body, at different times, the corresponding gel matrix selectively reaches the extrusion cylinder 110 from the storage container through the adaptive valve 113 and the pipeline 118, and the controllable power source 104 provides power according to the preset mode, and controls the piston 119 in the extrusion cylinder 11 to move through the piston shaft 120, so as to extrude the gel in the extrusion cylinder 110, and form a gel body through the controllable deformation extrusion head 106. The extruded gel body is finally in the cross-linking box 203, with the three-dimensional moving mechanism 202, accurately positioned and moved on the X, Y, and Z axes, and cross-linked and fixed by calcium chloride solution and ultraviolet light, and the sacrificial tube core, endothelial cell layer, pericyte layer, astrocyte layer and neuron layer are constructed point by point and layer by layer. After printing, the Pluronic F-127 gel tube core can be removed by a short period of low temperature of 10°C-15°C to complete the construction of the target vascularized neural tissue.

[0078] In the above-mentioned neural tissue construction process, the hydrogel material selection and the morphological size of the gel body are not fixed, but can be appropriately changed, and bioactive factors can be added as needed to induce angiogenesis and regulate neural activity, etc. The process method for three-dimensional tissue construction proposed in the present invention emphasizes the real-time controllability of the size and shape of the gel body unit, and can also control the number and density of cells in the cell clusters in the three-dimensional tissue, which provides support for the accurate simulation of complex tissue structures. It is possible to integrate multiple types of cells and bioactive factors in the same printing process and integrate the preparation and printing of gel bodies, which enhances the functionality and biomimetic properties of the tissue and improves the printing efficiency and controllability.

[0079] The printed multi-layered tissues with different structures are mainly divided into two parts: the hydrogel part that fills the gap and the gel bodies with different materials, different cells / bioactive factors, and different shapes and sizes. They have the characteristics of multi-cell integration, diverse materials, structural heterogeneity, precise three-dimensional construction, good bioactivity and customizable design. Figure 6 Figure 7 For example, in vascularized neural tissue, a multi-layered tissue is constructed by using endothelial cell-carrying gel bodies 301, pericyte-carrying gel bodies 302, astrocyte-carrying gel bodies 303, neuron-carrying gel bodies 304, sacrificial cavity gel bodies 305, and gap-filling gel 306, wherein the cell-carrying gel bodies mainly include two parts: a supporting gel 307 and a carried cell cluster 308.

[0080] The above contents are only for explaining the technical idea of ​​the present invention and cannot be used to limit the protection scope of the present invention. Any changes made on the basis of the technical solution in accordance with the technical idea proposed by the present invention shall fall within the protection scope of the claims of the present invention.

Claims

1. A multi-scale controllable three-dimensional tissue construction device, characterized in that: It comprises a gel body preparation unit (1), a three-dimensional mobile platform printing unit (2) and an integrated control terminal (3); The gel body preparation unit (1) comprises a controllable power source (104), a gel storage unit and a hydrogel extrusion element; The controllable power source (104) is used to provide the power required for extruding the gel and to control the extrusion amount and extrusion speed of the hydrogel; the gel storage unit is used to store a variety of hydrogel matrices carrying different types of cells or bioactive factors; the hydrogel extrusion element is used to extrude the hydrogel matrix to form a gel body; The integrated control terminal (3) is used to integrally control the output of the controllable power source (104), the movement of the gel storage unit, the movement of the hydrogel extrusion element and the movement of the three-dimensional mobile platform printing unit (2), thereby ensuring the accuracy and coordination of the entire printing process through global integrated control.

2. A multi-scale controllable three-dimensional tissue construction device according to claim 1, characterized in that: The gel storage unit comprises a plurality of gel storage containers (109) and a disc-type gel storage mechanism (105); The plurality of gel storage containers (109) are used to independently store different types of hydrogels; the plurality of gel storage containers (109) are fixed on a disc of a disc-type gel storage mechanism (105), and are driven by the disc-type gel storage mechanism (105) to perform circular motion and z-direction motion around a central axis.

3. A multi-scale controllable three-dimensional tissue construction device according to claim 2, characterized in that: The gel storage container (109) comprises a liquid inlet (112) and a liquid outlet; The gel storage container (109) is provided with a temperature sensing and regulating element (113), a stirring rod (114), a vibrator (115) and a capacity sensor (116). The temperature sensing and regulating element (113) can be wirelessly controlled to maintain a suitable temperature environment required for cell survival, thereby maintaining cell activity and gel stability. The stirring rod (114) and the vibrator (115) are used to stir and vibrate the hydrogel cell suspension; the capacity sensor (116) is used to monitor the amount of gel in the container in real time to ensure accurate control of the required amount of material during the printing process.

4. A multi-scale controllable three-dimensional tissue construction device according to claim 3, characterized in that: The liquid outlet of the gel storage container (109) is controlled by a conical adaptive valve (117) composed of a plurality of elastic sealing sheets. The adaptive valve (117) is subjected to the combined effect of the gravity of the liquid and the elastic sealing sheets to ensure that the liquid outlet can maintain a sealed state. When the disc-type gel storage mechanism (105) moves and the pipe (118) below is aligned and in contact with the liquid outlet, the top of the pipe (118) can push open the elastic sealing sheet to allow liquid to flow out. The flow rate is controlled by the contact degree between the top of the pipe (118) and the elastic sheet in the z direction. Once the mechanism moves away from the pipe (118), the elastic sealing sheet undergoes elastic deformation to automatically restore the closed state and close the liquid outlet.

5. The multi-scale controllable three-dimensional tissue construction device according to claim 4, characterized in that: The hydrogel extrusion element comprises an extrusion cylinder (110), an extrusion cylinder (111) and a controllably deformable extrusion head (106); One side of the extrusion barrel (110) is connected to the outlet of the pipeline (118), and the hydrogel enters the extrusion barrel (110) through the pipeline (118); the extrusion barrel (111) is located at the upper end of the extrusion barrel (110) and is used to apply pressure to the gel; the controllably deformable extrusion head (106) is connected to the end of the extrusion barrel (110), and the gel bodies are extruded and formed through it; The extrusion barrel (111) includes a piston (119) and a piston shaft (120), and pressure is generated by the movement of the piston (119) to squeeze the hydrogel matrix from the extrusion barrel to the controllable deformation extrusion head (106); the extrusion barrel also includes a pressure controller (121), including a pressure sensor and a pressure regulating component, which can monitor the pressure changes inside the extrusion barrel (111) in real time and control the pressure based on the hydrogel characteristics and printing requirements.

6. The multi-scale controllable three-dimensional tissue construction device according to claim 5, characterized in that: The controllable deformable extrusion head (106) includes a flexible inner wall (122) that can be stretched and deformed; a plurality of deformation drivers (123) connected to the flexible inner wall (122) that can be pulled outward and retracted inward, thereby completing a precise size and shape; a plurality of movable arms (124) respectively connected to the deformation drivers (123) to provide fixation and drive the deformation drivers (123) to move to assist them in controlling the size and shape of the extrusion head; an extrusion head controller (107) for controlling the movement of the deformation driver (123) and the movable arm (124) to ensure that the extrusion head is deformed in a predetermined mode; a flexible stop valve (108) located at the end of the controllable deformable extrusion head (106) that can be deformed along with the flexible inner wall (122) and is used to cut off the gel liquid flow to finally form a gel body carrying a cell cluster.

7. The multi-scale controllable three-dimensional tissue construction device according to claim 6, characterized in that: The three-dimensional mobile platform printing unit (2) comprises a printing platform (201) and a three-dimensional mobile mechanism (202); the three-dimensional mobile mechanism (202) is used to control the precise positioning and accurate movement of the printing platform (201) in a three-dimensional space.

8. The multi-scale controllable three-dimensional tissue construction device according to claim 7, characterized in that: The printing platform (201) is provided with a cross-linking box (203), which includes a plurality of chambers loaded with different liquid cross-linking agents to help the extruded gel bodies to be cross-linked and fixed; The four walls of the cross-linking box (203) are provided with ultraviolet light generating devices (204) with adjustable illumination direction, which are used for photo-cross-linking of the hydrogel; A temperature control layer is provided under the surface layer of the cross-linking box (203), which is used for thermal cross-linking of the hydrogel and can also ensure that the temperature is appropriate during the printing process.

9. The multi-scale controllable three-dimensional tissue construction device according to claim 8, characterized in that: The integrated control terminal (3) includes a model parser capable of reading and parsing an input three-dimensional tissue model and converting it into printing parameters; A user interface that allows users to edit and modify some of the 3D tissue model print code parameters, and also features a real-time preview window that displays the 3D tissue model and print path, allowing users to visualize the printing process; The control module generates detailed optimized printing instructions according to the parameters input by the user and the output of the model parser, and receives information transmitted by the capacity sensor (116), and finally controls and coordinates the operation of the controllable power source (104), the disc gel storage mechanism (105), the extruder head controller (107), the stop valve (108), the piston shaft (120), the pressure controller (121) and the three-dimensional moving mechanism (202); Data recording and tracking module records all data during the printing process.

10. A process for constructing a multi-scale controllable three-dimensional tissue, according to any one of claims 1 to 9, characterized in that: The following steps are involved: Step 1: Design a three-dimensional tissue model, clarify the purpose, expected function and biological requirements of the tissue model, and study and confirm the morphological characteristics of the target tissue, including size, shape, structural hierarchy and cell distribution; then determine the required cell type, design the distribution location and density of different cells in the tissue model, then determine the bioactive factor components that need to be integrated and their distribution location, and then select a suitable carrier hydrogel material to support cell growth and tissue function. Finally, the required tissue size and shape and the appropriate gel body size and shape need to be designed according to the cell morphology and gel properties; Step 2, preparing a gel matrix, mixing the selected cells and bioactive factors with a hydrogel material to prepare a biogel matrix with a suitable cell / bioactive factor density and storing it in a gel body preparation unit (1), specifically comprising: According to the needs of constructing tissues, the required cells and bioactive factors are mixed with hydrogel to prepare a gel matrix, and then the hydrogel matrix material is stored in multiple gel storage containers (109) from the liquid inlet (112). According to the cell type or active factor and hydrogel material, a temperature sensing and regulating element (113) is set to provide a suitable temperature to ensure cell activity and gel performance. If the hydrogel matrix needs to be stirred or vibrated, it can be selected to be loaded into a corresponding storage container and then processed accordingly; Step 3, importing the model and starting printing, converting the designed tissue model into a printing instruction through the integrated control terminal (3), and using it to modify and adjust the printing parameters, starting the three-dimensional mobile platform printing unit (2), and during the printing process, it is also necessary to use the cross-linking box (203) of the printing platform (20) to cross-link and shape the extruded gel bodies in real time to form the desired three-dimensional shape of the gel bodies. The specific process is: The experimenter needs to determine the tissue type and structure to be printed in advance according to the purpose and requirements, design the corresponding three-dimensional tissue model in steps, and then import the three-dimensional tissue model file into the integrated control terminal (3) to generate printing parameters and control instructions, including the circulation sequence volume and flow rate of the gel matrix and the corresponding pressure in the extrusion cylinder (111), the size and shape of the deformable extrusion head (106) and the output mode of the controllable power source (104). Then, confirm that the imported model is correct in the user interface and preview the printing path. The experimenter can modify or reset some parameters through the user interface of the integrated control terminal (3) according to his own needs and experience. Then, according to the hydrogel cross-linking requirements required for the printed tissue, add the corresponding cross-linking agent to the cross-linking box (203) or perform thermal cross-linking or use the ultraviolet light generating device (204) for ultraviolet cross-linking; Step 4, constructing three-dimensional tissues, with multi-type hydrogel matrices as the main body, using controllable deformation extrusion equipment to achieve precise control of the size and shape of the gel bodies, and cooperating with mobile devices, printing the gel bodies carrying cell clusters point by point and layer by layer, and directly and controllably constructing multi-cellular, multi-material, and multi-scale three-dimensional tissues from microscopic to macroscopic levels. The specific process is as follows: When the integrated control terminal (3) starts printing, according to the requirements of the constructed cell layer, at different times, the corresponding gel matrix selectively reaches the extrusion cylinder (110) from the storage container through the adaptive valve (113) and the pipeline (118), and the controllable power source (104) provides power according to the preset mode, and controls the movement of the piston (119) in the extrusion cylinder (11) through the piston shaft (120), so as to extrude the gel in the extrusion cylinder (110), and form gel bodies through the controllable deformation extrusion head (106). The extruded gel bodies are finally cross-linked and fixed in the cross-linking box (203), and then the three-dimensional moving mechanism (202) is precisely positioned and moved on the X, Y, and Z axes according to the design model, so as to realize the printing of the gel bodies one by one and layer by layer to finally form the desired tissue.

Images