High-density microsphere unit organ chip preparation device and preparation method thereof

Abstract

Compared with an existing organ chip construction method, the organ chip prepared by the organ chip construction method is low in resolution ratio and poor in separation performance, concentration gradient is difficult to set, and local experimental analysis of the organ chip is also difficult to carry out. Aiming at the defects in the prior art, the invention provides a preparation device and a preparation method of a high-density microsphere unit organ chip. Hydrogel (or cell suspension) and bio-oil are jointly injected into a 3D printing nozzle, on the basis of the static instability principle of two-phase fluid under the Laplace pressure, turbulent state transition occurs when the two-phase fluid crosses a micro-stepped structure, and the hydrogel (or cell suspension) clamped by the bio-oil is dispersed into high-density and uniform-size microspheres. The microspheres are stacked and solidified under the irradiation of a UV light source to form an organ model with an internal micro-channel. The problems that in an existing organ chip 3D printing technology, operation is complex, the production cost is high, the microsphere density is low, the microsphere monodispersity is poor, and cell density distribution is difficult to control are solved.

Description

Technical Field

[0001] The present invention relates to the field of organ chip preparation and processing, and in particular to a microfluidic device and a preparation method capable of preparing a high-density microsphere unit organ chip. Background Art

[0002] Organ-on-a-chip is a new interdisciplinary biomedical manufacturing technology that utilizes microfluidic chip technology combined with multidisciplinary knowledge such as cell biology and materials science to simulate and construct miniaturized and functional human or experimental animal organ models in vitro. It is a microfluidic cell culture device that can simulate the in vivo multicellular structure, tissue-tissue interface, physical and chemical microenvironment, and vascular perfusion.

[0003] Existing organ chip processing methods include photolithography, molding, laser etching, microcontact imprinting, 3D printing, etc. Among them, 3D printing is not only simple to operate and has high processing precision, but also can prepare three-dimensional complex structures, so it has been widely used. In the 3D printing method, the substance extruded by the printer nozzle is often a cell suspension or a hydrogel. By designing a suitable 3D model and selecting a suitable printing material (such as a polymer material with good biocompatibility), an organ chip with a complex microstructure can be directly printed. However, the organ chip prepared by the above method has poor separation, it is difficult to set a concentration gradient, and it is also difficult to conduct local experimental analysis of the organ chip.

[0004] In addition, a medical 3D printing controllable microsphere preparation device is disclosed in Publication No. CN117209806B, but the microsphere density is low, and the volume ratio (dispersed phase volume / dispersed phase volume+continuous phase volume) is less than 40%. The microsphere product needs to be precipitated, purified, collected, etc., so as to increase the volume ratio of the microsphere and re-inject it into the 3D printing device to complete the construction of the organ chip. The operation is very complicated. A cell microsphere 3D printing nozzle and printing method are disclosed in Publication No. CN118546784A. Although it can be used to print cell microspheres, the microspheres are uneven in size, poorly monodisperse, and the cells in each unit are not efficiently separated. Summary of the invention

[0005] In view of the shortcomings of the prior art, the purpose of the present invention is to provide a high-density microsphere unit organ chip preparation device and preparation method. The present invention designs a 3D printing nozzle with a special structure, which contains a shallow channel and a deep channel. The micro-step structure between the two can transform the two-phase laminar flow into turbulent flow and realize the generation of high-density, monodisperse microspheres. During the printing process, the hydrogel (or cell suspension) and the bio-oil are injected into the 3D printing nozzle together, and the two form a sheath flow laminar state. Based on the static instability principle of the two-phase fluid under Laplace pressure, the turbulent state changes when crossing the micro-step structure, and the hydrogel (or cell suspension) sandwiched by the bio-oil is discretized into high-density, uniform-sized microspheres. Further, the microspheres are stacked and solidified under the irradiation of a UV light source to form an organ model with an internal microchannel. The above content solves the problems of complex operation, high production cost, low microsphere density, poor microsphere monodispersity, and difficult control of cell density distribution in the existing organ chip 3D printing technology.

[0006] In order to achieve the above object, the present invention is implemented through the following technical solutions:

[0007] First aspect The present invention provides a high-density microsphere unit organ chip preparation device, comprising:

[0008] The liquid storage device includes two liquid storage containers, one for storing a continuous phase fluid (generally bio-oil), and the other for storing a dispersed phase fluid (generally hydrogel, cell suspension).

[0009] The 3D printing system, including a computer control system and an XYZ three-coordinate displacement platform, allows users to input organ chip model files and key parameters of microspheres generated by the print head, such as size, density and arrangement. The system converts these parameters into printing instructions and monitors and adjusts them in real time during the printing process to ensure printing accuracy and quality.

[0010] The nozzle device (core device) used to produce microspheres is equipped with a shallow channel connecting two liquid storage bottles, a cross microchannel that intersects the two-phase fluid to form a sheath flow, a micro-step structure, and a deep channel according to the fluid flow order. Through the fluid pipeline, the dispersed phase and continuous phase liquid storage bottles are pumped to the nozzle, and then the two-phase fluid generates high-density, monodisperse microspheres under the Rayleigh-Plateau static unstable hydrodynamic mode, and is stacked and solidified layer by layer under the drive of the 3D printing system, and finally formed into a microsphere unit organ chip.

[0011] The pressure pumping device is equipped with a controller and is connected to the liquid storage device to provide necessary power to drive the fluid flow.

[0012] As a further implementation, the pressure pumping system is a pressure pump, and the pressure pump is a vane pump, a positive displacement pump, or an electromagnetic pump.

[0013] As a further implementation, the continuous phase liquid storage bottle and the dispersed phase liquid storage bottle are connected to the continuous phase inlet and the dispersed phase inlet of the 3D printing system.

[0014] As a further implementation, the pressure pumping system can accurately control the pressure of the two-phase fluid in the 3D printing nozzle.

[0015] As a further implementation method, the dispersed phase fluid is an aqueous substance, and its components include but are not limited to cell suspension, hydrogel, polymer monomer, cross-linking agent, and initiator; the continuous phase fluid is an oily substance that is insoluble in aqueous substances, and its components include but are not limited to vegetable oil and surfactant.

[0016] Second aspect The present invention provides a method for preparing a microsphere unit organ chip, using the microsphere unit organ chip preparation system as described in the first aspect, comprising the following steps:

[0017] Specifically, the pressure pump delivers high-pressure gas to the dispersed phase storage bottle and the continuous phase storage bottle, and squeezes the two-phase fluid into the fluid hose. Among them, the controller is responsible for adjusting the air pressure reaching the storage bottle to ensure that the dispersed phase fluid and the continuous phase fluid can reach the printer nozzle at a suitable flow rate. The nozzle has microstructures such as shallow channels and deep channels. The continuous phase fluid and the dispersed phase fluid converge at the cross-intersection position of the shallow channel area and flow in a sheath flow state. The sheath flow moves forward and reaches the deep channel in the nozzle. When crossing the micro-step structure, the tiny disturbance between the two-phase interface of the sheath flow is sharply amplified, resulting in an instantaneous Laplace pressure difference. This pressure forces the dispersed phase fluid to neck, and then break and form microspheres of uniform size. In the above process, the dispersed phase breaks spontaneously under the action of static instability, without the shearing action of the continuous phase, so high-density dispersed phase microspheres are obtained, and its volume share can reach up to 92%. The extremely high volume share avoids the sedimentation, purification, and re-injection of the microspheres. As the pressure pumping system continues to operate, the two-phase fluid is continuously injected into the nozzle device, and microspheres are continuously generated from the nozzle. Driven by the 3D printing system, the nozzle moves along a predetermined three-dimensional trajectory and solidifies under ultraviolet light, completing the stacking of layers and ultimately constructing a microsphere unit organ chip.

[0018] As a further implementation, the controller may control the unit flow rates of the dispersed phase fluid and the continuous phase fluid, and control the flow rates of the dispersed phase fluid and the continuous phase fluid to be in an appropriate ratio by controlling the pressure of the driving gas.

[0019] As a further implementation method, the dispersed phase fluid and the continuous phase fluid are squeezed by the high-pressure gas, and the dispersed phase fluid and the continuous phase fluid are respectively transported to the dispersed phase inlet and the continuous phase inlet of the 3D printing system through the liquid pipeline.

[0020] As a further implementation method, the necked and fractured dispersed phase fluid enters the deep channel and forms microspheres under the principle of minimum surface energy.

[0021] As a further implementation method, when the pressure of the continuous phase storage bottle is maintained at the same, the greater the pressure of the dispersed phase fluid storage bottle, the larger the size of the generated microspheres, showing a positive correlation.

[0022] As a further implementation method, when the flow rate of the continuous phase fluid is maintained at the same, the greater the flow rate of the dispersed phase fluid, the larger the size of the generated hydrogel microspheres, showing a positive correlation.

[0023] As a further implementation, other pressures of the pressure pumping system are 0 to 100 kPa, and the pressure adjustment range of the controller is 0 to 100 kPa.

[0024] The beneficial effects of the present invention are as follows:

[0025] Based on the Rayleigh-Plateau static instability of the two-phase fluid interface, the present invention designs a micro-step structure composed of shallow channels and deep channels in the 3D printing nozzle. The structure of the nozzle is completely different from that of the traditional 3D printing nozzle. The micro-step structure inside the nozzle can induce drastic changes in the dispersed phase-continuous phase two-phase fluid interface, forcing the dispersed phase fluid to periodically neck and break to form high-volume-share, monodisperse microspheres, thereby realizing the direct construction of microsphere unit organ chips without the precursor of microsphere sedimentation, purification, and re-injection operations, greatly improving the molding efficiency and molding quality of the microsphere unit organ chips. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0027] Figure 1 It is a schematic structural diagram of a high-density microsphere unit organ chip preparation device in Example 1 of the present invention.

[0028] Figure 2 The first embodiment is to adopt Figure 1 Schematic diagram of the high-density, monodisperse microspheres produced by the device. The microspheres are densely distributed in the organ chip in a stacked manner.

[0029] Figure 3 The first embodiment is to adopt Figure 1The organ chip constructed by the device (from left to right, the arterial organ chip in three states).

[0030] Figure 4 The first embodiment is to adopt Figure 1 From the enlarged partial view of the organ chip constructed by the device, it can be seen that the microspheres in the organ chip have high density and good monodispersity, and can be stacked layer by layer and connected into a whole.

[0031] Figure 5 It is a graph of the gas pressure of the continuous phase fluid and the dispersed phase fluid in Example 2 of the present invention, and the organ chip microsphere unit generated under this condition.

[0032] Figure 6 This is a graph of the atmospheric pressures of the continuous phase fluid and the dispersed phase fluid in Example 3 of the present invention, and the organ chip microsphere unit generated under this condition.

[0033] Figure 7 1 is a flow rate curve diagram of the continuous phase fluid and the dispersed phase fluid in Example 4 of the present invention, and the organ chip microsphere unit generated under this condition.

[0034] Figure 8 It is a flow rate curve diagram of the continuous phase fluid and the dispersed phase fluid in Example 5 of the present invention, and the organ chip microsphere unit generated under this condition.

[0035] In the figure: the distances or sizes between parts are exaggerated to show the positions of various parts, and the schematic diagram is for reference only.

[0036] Among them: 1. Pressure pump; 2. Controller; 3. Gas pipeline; 4. Dispersed phase storage bottle; 5. Continuous phase storage bottle; 6. Dispersed phase fluid; 7. Continuous phase fluid; 8. Fluid pipeline; 9. 3D printing system; 10. Computer control system; 11. Nozzle containing micro-step structure; 12. Microsphere; 13. Shallow channel; 14. Deep channel; 15. Micro-step; 16. Sheath flow. DETAILED DESCRIPTION

[0037] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs.

[0038] Embodiment 1

[0039] In a typical embodiment of the present invention, reference Figure 1 As shown, a high-density microsphere unit organ chip preparation device includes a pressure pump device 1, a pressure / flow controller device 2, liquid storage devices 4 and 5, a 3D printing system 9, and a computer control system 10.

[0040] The pressure pump device 1 is connected to the pressure / flow controller device 2, and then connected to the dispersed phase liquid storage bottle 4 and the continuous phase liquid storage bottle 5, providing the necessary power to drive the flow of the fluid. The pressure pump device 1 is a vane pump or a volumetric pump or an electromagnetic pump to adapt to different fluid properties and pressure requirements.

[0041] The liquid storage devices 4 and 5 are composed of two liquid storage containers: one is a continuous phase liquid storage bottle 5 for storing a continuous phase fluid 7, and the other is a dispersed phase liquid storage bottle 4 for storing a dispersed phase fluid 6. The pressure / flow controller device 2 controls the continuous phase liquid storage bottle 5 and the dispersed phase liquid storage bottle 4 through the gas pipeline 3 to ensure a stable supply of the fluid.

[0042] The 3D printing nozzle device 11 is provided with a shallow channel 11 connecting the continuous phase storage bottle 5 and the dispersed phase storage bottle 4, a cross microchannel for joining the two phases of fluid to form a sheath flow 13, a micro-step structure, and a deep channel 12 in the order of fluid flow. Through the fluid pipeline 8, the continuous phase fluid 7 and the dispersed phase fluid 6 are accurately pumped into the high-precision 3D printing nozzle 11. In the 3D printing nozzle 11, the two phases of fluid meet at the cross microchannel position and form a sheath flow 16, which is then disturbed when crossing the micro-step structure 15; at this time, the dispersed phase fluid 6 breaks into microspheres 12.

[0043] The 3D printing system 9 is used to drive the three-dimensional movement of the 3D printing nozzle device 11, and the three-dimensional movement path is the organ chip construction path.

[0044] The computer control system 15 is connected to the pressure / flow controller device 2 and the 3D printing system 9, allowing the user to input key parameters of the microspheres, such as size, density and arrangement. The computer control system converts these parameters into printing instructions and performs real-time monitoring and adjustment during the printing process to ensure printing accuracy and quality.

[0045] The gas pipeline 3 and the fluid pipeline 8 are made of glass, silicone or plastic.

[0046] In this embodiment, a pressure pump 1 is used to produce high-pressure gas and transport it to the dispersed phase liquid storage bottle 4 and the continuous phase liquid storage bottle 5, and the two-phase fluid is squeezed into the fluid hose. Among them, the controller 2 is responsible for adjusting the air pressure reaching the liquid storage bottle to ensure that the dispersed phase fluid 6 and the continuous phase fluid 7 can reach the printer nozzle 11 at a suitable flow rate. The nozzle 11 has microstructures such as shallow channels 13 and deep channels 14. The continuous phase fluid 7 and the dispersed phase fluid 6 converge at the cross position of the shallow channel area and flow in a sheath flow state 16. The sheath flow 16 moves forward and reaches the deep channel 14 in the nozzle. When crossing the micro-step structure 15, the tiny disturbance between the two-phase interface of the sheath flow 16 is sharply amplified, resulting in an instantaneous Laplace pressure difference, which forces the dispersed phase fluid to neck, and then break and form microspheres 12 of uniform size. In the above process, the dispersed phase spontaneously breaks under the action of static instability, without the need for shearing of the continuous phase, thus obtaining high-density dispersed phase microspheres 12, whose volume share can reach up to 92%. The extremely high volume share avoids the sedimentation, purification, and re-injection of the microspheres. With the continuous operation of the pressure pumping system, the two-phase fluid is continuously injected into the nozzle device, and the microspheres 12 are continuously generated from the nozzle 11. Driven by the 3D printing system 9, the nozzle 11 moves according to a predetermined three-dimensional trajectory and solidifies under ultraviolet light, thereby completing the stacking of layers and finally constructing a microsphere unit organ chip.

[0047] In this embodiment, based on the Rayleigh-Platte instability of the two-phase fluid interface, a micro-step structure 15 consisting of a shallow channel 13 and a deep channel 14 is designed in the 3D printing nozzle 11. The nozzle 11 is completely different from the structure of a traditional 3D printing nozzle. The micro-step structure 15 inside it can induce drastic changes in the dispersed phase-continuous phase two-phase fluid interface, forcing the dispersed phase fluid 6 to periodically neck and break, forming high volume-occupying, monodisperse microspheres 12, thereby realizing the direct construction of the microsphere unit organ chip without the precursor of microsphere sedimentation, purification, and re-injection operations, greatly improving the molding efficiency and molding quality of the microsphere unit organ chip.

[0048] The gas pressure in the pressure pump 1 is 0 to 100 kPa, and a variety of preset pressure options are provided, such as 20 kPa, 50 kPa, 70 kPa, 80 kPa or 100 kPa, but are not limited to the listed values. Other unlisted values ​​within the range are also applicable to meet different printing requirements.

[0049] The pressure adjustment range of the controller 2 is 0 to 100 kPa, such as 20 kPa, 40 kPa, 60 kPa, 80 kPa or 100 kPa, but is not limited to the listed values, and other values ​​not listed within the range are also applicable.

[0050] Using the device described in this embodiment, the generation frequency of the microspheres 12 is 10 Hz to 5000 Hz, such as 10 Hz, 500 Hz, 1000 Hz or 1500 Hz, but is not limited to the listed values, and other values ​​not listed within the numerical range are also applicable.

[0051] The flow rate of the sheath flow 16 ranges from 10 μl / min to 5500 μl / min, including 20 μl / min, 300 μl / min, 1500 μl / min or 3500 μl / min, but is not limited to the listed values, and other unlisted values ​​within the numerical range are also applicable.

[0052] The shape design of the organ chip model composed of microspheres 12 is not limited to three-dimensional graphics such as cylinders, cones, cubes, etc. The system supports the input of different 3D model files to create a variety of model shapes to meet personalized printing needs.

[0053] Figure 2 The plan views of hydrogel microspheres generated at different printing levels are shown. Specifically, the diagrams show the distribution of microspheres from the first to the sixth layers, and the microspheres in each layer are precisely arranged according to the predetermined design parameters to form a uniform and orderly planar structure. These images not only show the distribution density and arrangement pattern of the microspheres in each layer, but also reflect the consistency and repeatability of the printing process.

[0054] Figure 3 The three-dimensional structures of hydrogel microspheres formed under single-layer, three-layer and multi-layer printing conditions are shown. Each layer of microspheres is achieved by precisely controlling the printing parameters to construct three-dimensional structures with different complexities. These structural diagrams not only show the stacking effect of microspheres at different numbers of layers, but also highlight the potential of the device in constructing complex three-dimensional biomaterial structures.

[0055] Figure 4 Microscopic images of hydrogel microspheres observed at different scales are shown. The images clearly show the morphology, surface features and size consistency of the microspheres, confirming that the printing process can accurately control the microstructure of the microspheres. The above microscopic images are an important basis for evaluating the printing quality, microsphere uniformity and size accuracy.

[0056] Embodiment 2

[0057] This embodiment provides a method for preparing a high-density microsphere unit organ chip, using the high-density microsphere unit organ chip preparation device as described in Example 1, specifically comprising the following steps:

[0058] (1) The pressure pump 1 compresses the external air and delivers it to the controller 2;

[0059] (2) The controller 2 adjusts the gas pressure and delivers 600 kPa and 1000 kPa compressed gas to the dispersed phase storage bottle 4 and the continuous phase storage bottle 5 respectively through the gas hose 3 (e.g. Figure 5 shown);

[0060] (3) The dispersed phase fluid 6 and the continuous phase fluid 7 are squeezed by the high-pressure gas, and the two-phase fluids are respectively transported to the dispersed phase inlet and the continuous phase inlet in the 3D printing nozzle 11 through the fluid pipeline 8, and then enter the shallow channel 13;

[0061] (4) the dispersed phase fluid 6 and the continuous phase fluid 7 meet at a cross position in the shallow channel 13 and form a sheath flow 16;

[0062] (5) When the sheath flow 16 crosses the micro-step structure 15 and enters the deep channel 14, the spatial mutation causes the disturbance of the sheath flow 16 on the two-phase fluid interface to be instantly amplified, thereby increasing the change in the interface curvature, that is, the Laplace pressure difference on both sides of the interface increases; the Laplace stress causes the interface curvature to be further enhanced, and the dispersed phase fluid 6 is squeezed by the continuous phase fluid 7 on both sides to form a contraction neck;

[0063] (6) The contraction neck is broken, and the broken dispersed phase fluid 6 enters the deep channel 14 and forms the organ chip microsphere unit 12 under the principle of minimum surface energy;

[0064] (7) Steps (5) and (6) are repeated alternately, and the sheath fluid 16 body retracts into the shallow channel 13, and under the drive of the pressure source, the next microsphere generation process begins. The macroscopic manifestation is the high-frequency oscillation of the dispersed phase fluid 6 at the position of the micro-step 15, and the continuous ejection of the organ chip microsphere unit 12.

[0065] (8) The computer control system 10 controls the 3D printing system according to the input organ chip 3D model file, and further controls the 3D movement of the 3D printing nozzle 11. The nozzle 11 continuously generates high-density, monodisperse microspheres while performing 3D movement. The microspheres are solidified and stacked layer by layer to construct a microsphere unit organ chip.

[0066] Embodiment 3

[0067] This embodiment provides a method for preparing a high-density microsphere unit organ chip. The difference from the second embodiment is that the pressures of the dispersed phase liquid storage bottle 4 and the continuous phase gas storage bottle 5 are 800 kPa and 1000 kPa respectively (e.g. Figure 6 As shown in FIG. 1 ), the high-pressure gas pushes the dispersed phase gas 6 and the continuous phase fluid 7 to form a sheath flow 16. Under the continuous drive of the high-pressure gas, the organ chip microsphere unit 12 (as shown in FIG. 1 ) is generated. Figure 6 shown).

[0068] Combination Figure 5 and Figure 6 It can be found that when the pressure of the continuous phase storage bottle 5 is maintained at the same, the greater the pressure of the dispersed phase storage bottle, the larger the size of the generated microsphere unit 12 is, showing a positive correlation.

[0069] Embodiment 4

[0070] This embodiment provides a method for preparing a high-density microsphere unit organ chip. The difference from the second and third embodiments is that under the regulation of the controller 2, the pressures of the dispersed phase liquid storage bottle 4 and the continuous phase gas storage bottle 5 are 3 μl / min and 9 μl / min respectively (e.g. Figure 7 As shown in FIG. 1 ), under this flow rate configuration, the dispersed phase gas 6 and the continuous phase fluid 7 form a sheath flow 16. Under the continuous drive of the high pressure gas, the organ chip microsphere unit 12 (as shown in FIG. 1 ) is formed. Figure 7 shown).

[0071] Embodiment 5

[0072] This embodiment provides a method for preparing a high-density microsphere unit organ chip. The difference from the second, third and fourth embodiments is that under the regulation of the controller 2, the pressures of the dispersed phase liquid storage bottle 4 and the continuous phase gas storage bottle 5 are 6 μl / min and 9 μl / min respectively (e.g. Figure 8 As shown in FIG. 1 ), under this flow rate configuration, the dispersed phase gas 6 and the continuous phase fluid 7 form a sheath flow 16. Under the continuous drive of the high pressure gas, the organ chip microsphere unit 12 (as shown in FIG. 1 ) is formed. Figure 8 shown).

[0073] Combination Figure 7 and Figure 8 It can be found that when the pressure in the continuous phase storage bottle 5 is maintained at the same level, the greater the flow rate of the dispersed phase fluid 6, the larger the size of the generated microspheres 12, showing a positive correlation.

[0074] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A high-density microsphere unit organ chip preparation device, characterized in that: include: A liquid storage device, comprising two liquid storage containers, one for storing a continuous phase fluid (generally bio-oil), and the other for storing a dispersed phase fluid (generally hydrogel, cell suspension); The 3D printing system includes a computer control system and an XYZ three-coordinate displacement platform, which allows users to input organ chip model files and key parameters of microspheres generated by the print head, such as size, density, and arrangement. The system converts these parameters into printing instructions and conducts real-time monitoring and adjustments during the printing process to ensure printing accuracy and quality; The nozzle device (core device) used to produce microspheres is equipped with a shallow channel connecting two liquid storage bottles, a cross microchannel that intersects the two-phase fluid to form a sheath flow, a micro-step structure, and a deep channel according to the fluid flow order. Through the fluid pipeline, the dispersed phase and continuous phase liquid storage bottles are pumped to the nozzle, and then the two-phase fluid generates high-density, monodisperse microspheres under the Rayleigh-Plateau static unstable hydrodynamic mode, and is stacked and solidified layer by layer under the drive of the 3D printing system, and finally formed into a microsphere unit organ chip; The pressure pumping device is equipped with a controller and is connected to the liquid storage device to provide necessary power to drive the fluid flow.

2. A high-density microsphere unit organ chip preparation device according to claim 1, characterized in that: The pressure pumping device is a pressure pump, and the pressure pump is a vane pump, a positive displacement pump, or an electromagnetic pump.

3. The high-density microsphere unit organ chip preparation device according to claim 1, characterized in that: The continuous phase liquid storage bottle and the dispersed phase liquid storage bottle are connected to the continuous phase inlet and the dispersed phase inlet of the 3D printing nozzle.

4. The high-density microsphere unit organ chip preparation device according to claim 1, characterized in that: The pressure pumping system can accurately control the pressure of the two-phase fluid in the 3D printing nozzle.

5. The high-density microsphere unit organ chip preparation device according to claim 1, characterized in that: The dispersed phase fluid is an aqueous substance, and its components include but are not limited to cell suspension, hydrogel, polymer monomer, cross-linking agent, and initiator; the continuous phase fluid is an oily substance that is insoluble in aqueous substances, and its components include but are not limited to vegetable oil and surfactant.

6. A method for preparing a high-density microsphere unit organ chip, using the high-density microsphere unit organ chip preparation device according to any one of claims 1 to 3, characterized in that: The following steps are involved: The pressure pump delivers high-pressure gas to the dispersed phase storage bottle and the continuous phase storage bottle, and squeezes the two-phase fluid into the fluid hose; the controller is responsible for adjusting the gas pressure reaching the storage bottle to ensure that the dispersed phase fluid and the continuous phase fluid can reach the printer nozzle at a suitable flow rate; the nozzle has microstructures such as shallow channels and deep channels, and the continuous phase fluid and the dispersed phase fluid converge at the cross position of the shallow channel area and flow in coordination in a sheath flow state; the sheath flow moves forward and reaches the deep channel in the nozzle. When crossing the micro-step structure, the tiny disturbance between the two-phase interface of the sheath flow is sharply amplified, resulting in an instantaneous Laplace pressure difference, which forces the dispersed phase The fluid necks, breaks and forms microspheres of uniform size. In the above process, the dispersed phase breaks spontaneously under the action of static instability, without the need for shearing of the continuous phase. As a result, high-density dispersed phase microspheres are obtained, with a volume share of up to 92%. The extremely high volume share avoids the sedimentation, purification and re-injection of the microspheres. With the continuous operation of the pressure pumping system, the two-phase fluid is continuously injected into the nozzle device, and microspheres are continuously generated from the nozzle. Driven by the 3D printing system, the nozzle moves according to a predetermined three-dimensional trajectory and solidifies under ultraviolet light, thereby completing the stacking of layers and finally constructing a microsphere unit organ chip.

7. The method for preparing a high-density microsphere unit organ chip according to claim 6, characterized in that: The controller can control the unit flow rate of the dispersed phase fluid and the continuous phase fluid, and by controlling the pressure of the driving gas, the flow rate of the dispersed phase and the flow rate of the continuous phase fluid are controlled to be in an appropriate ratio.

8. The method for preparing a high-density microsphere unit organ chip according to claim 6, characterized in that: The dispersed phase fluid and the continuous phase fluid are squeezed by the high-pressure gas, and the dispersed phase fluid and the continuous phase fluid are respectively transported to the dispersed phase inlet and the continuous phase inlet of the 3D printing system through the liquid pipeline.

9. The method for preparing a high-density microsphere unit organ chip according to claim 6, characterized in that: The necked and fractured dispersed phase fluid enters the deep channel and forms microspheres under the principle of minimum surface energy.

10. The method for preparing a high-density microsphere unit organ chip according to claim 6, characterized in that: When the pressure in the continuous phase reservoir bottle remains the same, the greater the pressure in the dispersed phase fluid reservoir bottle, the larger the size of the generated microspheres, showing a positive correlation.

11. The method for preparing a high-density microsphere unit organ chip according to claim 6, characterized in that: When the flow rate of the continuous phase fluid remains the same, the greater the flow rate of the dispersed phase fluid, the larger the size of the generated hydrogel microspheres, showing a positive correlation.

12. The method for preparing a high-density microsphere unit organ chip according to claim 6, characterized in that: The other pressures of the pressure pumping system are 0~100kPa, and the pressure adjustment range of the controller is 0~100kPa.

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