A sedimentation bioprinting device and printing method for ultra-high cell density functional organs mediated by acoustic fluid

Abstract

The present invention discloses a sedimentation-type bioprinting device and a printing method of an ultra-high cell density functional organ mediated by acoustic fluid, which belongs to the field of medical engineering manufacturing technology. The device includes an array surface acoustic wave chip, a printing container, a printing platform and a curing light source; the printing container is used to hold printing ink; the printing platform is horizontally arranged in the printing container and can move up and down in the printing ink; the array surface acoustic wave chip and the curing light source are both located above the printing container; the array surface acoustic wave chip includes a plurality of interdigital electrodes arranged in a circular ring in the same horizontal plane; the central axis of each interdigital electrode deviates from its direction toward the center of the circle; the array surface acoustic wave chip contacts the printing ink and can form a focused acoustic flow in the printing ink between it and the printing platform to gather cells in the printing ink; the curing light source can be projected into the printing ink above the printing platform and solidify it. The present invention has the advantages of fast printing and adjustable cell concentration.

Description

Technical Field

[0001] The present invention belongs to the field of medical manufacturing technology, and relates to a bioprinting device, and in particular to an acoustic fluid-mediated sedimentation bioprinting device for ultra-high cell density functional organs and a printing method thereof. Background Art

[0002] 3D bioprinting technology is a new technology that uses computer three-dimensional models as "drawings" and assembles special "bio-inks" to ultimately create artificial organs and biomedical products. With the rapid development of related technologies, 3D bioprinting also has broad application prospects in economic life, national defense and military fields.

[0003] A 3D bioprinter is a device that can position and assemble biomaterials or cell units according to the principle of additive manufacturing under the drive of a digital three-dimensional model to manufacture medical devices, tissue engineering scaffolds, tissue organs and other products. The principle of biological 3D printing technology is to accurately position cells and biomaterials for printing, and ultimately form human organs that meet anatomical structure and functional requirements. Its basic workflow includes the application of medical imaging technology, the use of computer-aided design software, the printing of biomaterials and cells, and the cultivation of cells and the maturation of organs. Through this process, the regeneration and transplantation of human organs can be achieved.

[0004] Although some bioprinting methods or devices have emerged, they cannot achieve ultra-high cell density printing compared to natural tissues where the cell density is generally greater than 40 million cells per milliliter. This is because in jet-based bioprinting, mechanical extrusion or traction of high-cell-density bio-inks usually causes cell damage, while in lithography-based bioprinting, there is a lack of effective and controllable cell assembly strategies. These bottlenecks limit the application of traditional 3D bioprinting. Summary of the invention

[0005] The present invention provides an acoustic fluid-mediated sedimentation-type bioprinting device for ultra-high cell density functional organs and a printing method thereof, so as to overcome the defects of the prior art.

[0006] To achieve the above-mentioned purpose, the present invention provides a sedimentation-type bioprinting device for ultra-high cell density functional organs mediated by acoustic fluid, which has the following characteristics: it comprises an array surface acoustic wave chip, a printing container, a printing platform and a curing light source; the printing container is used to hold printing ink, and the printing ink is a mixture of cells and photocurable biological ink; the printing platform is horizontally arranged in the printing container and can move up and down in the printing ink; the array surface acoustic wave chip and the curing light source are both fixedly located above the printing container; the array surface acoustic wave chip comprises a plurality of forked The invention relates to a method for preparing an array of surface acoustic wave chips, wherein the forked electrodes are focusing surface acoustic wave forked electrodes; the central axis of each forked electrode deviates from the direction toward the center of a circle (i.e., deviates counterclockwise from the line connecting the forked electrode and the center of the circle), and the deviation directions of the multiple forked electrodes are the same, which are all clockwise or counterclockwise; the array surface acoustic wave chip is in contact with the printing ink (it can be in contact with the liquid surface of the printing ink or can be extended into the printing ink to achieve contact), and can form a focused acoustic flow in the printing ink between the array surface acoustic wave chip and the printing platform to gather cells in the printing ink; the curing light source can be projected into the printing ink above the printing platform and solidify it.

[0007] Furthermore, the present invention provides an acoustic fluid-mediated sedimentation bioprinting device for ultra-high cell density functional organs, which may also have the following characteristics: wherein, in the array surface acoustic wave chip, the deflection angles of multiple interdigitated electrodes are the same.

[0008] Furthermore, the present invention provides an acoustic fluid-mediated sedimentation bioprinting device for ultra-high cell density functional organs, which may also have the following characteristics: wherein the deviation angle of the interdigitated electrodes is: 0°<deviation angle<90°.

[0009] Furthermore, the present invention provides a sedimentation-type bioprinting device for ultra-high cell density functional organs mediated by acoustic fluid, which may also have the following characteristics: wherein, in the array surface acoustic wave chip, a plurality of interdigital electrodes are evenly distributed on a ring, and the plurality of interdigital electrodes share the same piezoelectric substrate, that is, a plurality of electrode patterns are processed on one piezoelectric substrate.

[0010] The production of array surface acoustic wave chips is based on semiconductor preparation technology. First, the electrode arrangement is designed using computer-aided software, and then the metal electrodes are processed on the piezoelectric substrate using standard soft lithography and electron beam metal deposition technology. The specific method is: a mask with electrode patterns of multiple interdigitated electrodes is obtained by thermoplastic molding, positive photoresist is spin-coated on the lithium niobate piezoelectric substrate, and the electrode pattern on the mask is obtained by replication and elution; then chromium film and gold film are deposited on the piezoelectric substrate using electron beam evaporation; finally, the array surface acoustic wave chip is eluted in formaldehyde at 60°C.

[0011] Furthermore, the present invention provides an acoustic fluid-mediated sedimentation bioprinting device for ultra-high cell density functional organs, which may also have the following characteristics: wherein the number of electrode pairs in the interdigitated electrodes is 3 to 10 pairs, and the wavelength is 200 to 350 μm. The driving signal of the array surface acoustic wave chip is a sinusoidal alternating current input with a voltage of 250 mVpp.

[0012] Furthermore, the present invention provides an acoustic fluid-mediated sedimentation bioprinting device for ultra-high cell density functional organs, which may also have the following characteristics: wherein the printing container can move up and down.

[0013] Furthermore, the present invention provides an acoustic fluid-mediated sedimentation-type bioprinting device for ultra-high cell density functional organs, which may also have the following characteristics: wherein the device also includes a print head; the array surface acoustic wave chip is fixed at the bottom of the print head, specifically the piezoelectric substrate is fixed to the print head, and the electrode pattern (gold electrode) faces the printing ink.

[0014] The present invention also provides a printing method of a sedimentation-type bioprinting device for ultra-high cell density functional organs mediated by acoustic fluid, which has the following characteristics: the printing method of a single-layer tissue organ comprises the following steps: S1.1, exposing a circular projection through the curing light source to solidify the printing ink to form a circular sound field boundary; S1.2, turning on the array surface acoustic wave chip to form a focused surface acoustic wave sound field, inducing a fluid vortex within the sound field boundary, thereby gathering cells in the printing ink; S1.3, exposing a projection of a shape to be printed through the curing light source, so that the printing ink is solidified into a tissue organ of the shape to be printed containing aggregated cells; S1.4, exposing a projection containing the shape of the printed tissue organ through the curing light source, so that the printing ink is solidified into a single-layer printed structure containing the tissue organ in step three.

[0015] Furthermore, the present invention provides a printing method of a sedimentation-type bioprinting device for ultra-high cell density functional organs mediated by acoustic fluid, which may also have the following characteristics: the printing method of a multi-layer tissue organ comprises the following steps: S1, first printing a single-layer printing structure according to the printing method of a single-layer tissue organ; S2, moving the printing platform downward, repeating S1.1 to S1.4, and printing a second-layer printing structure above the single-layer printing structure; S3, repeating S2 n times to obtain an n+2-layer printing structure, where n≥0.

[0016] Furthermore, the present invention provides a printing method of a sedimentation-type bioprinting device mediated by acoustic fluid for ultra-high cell density functional organs, which may also have the following characteristics: wherein, the distance between the lower surface of the array surface acoustic wave chip / previous layer printing structure and the printing platform is the thickness of the layer of printing structure.

[0017] The beneficial effects of the present invention are as follows: the present invention provides an acoustic fluid-mediated sedimentation bioprinting device and a printing method for ultra-high cell density functional organs, which are used for three-dimensional printing of bionic organs with ultra-high cell density and complex geometric structures and functions. The method is based on the vortex caused by focusing surface acoustic waves to gather cells on the pre-polymerized solid-liquid interface, thereby obtaining a cell hydrogel carrier with a cell density equivalent to that of natural tissue. Then, by integrating a sedimentation stereolithography process, a bionic cell structure with three-dimensional complex layers can be generated. In addition, the high cell density makes the cell tissue model present more mature functions. The cell aggregation induced by sound waves can greatly increase the cell concentration in the printing ink, thereby realizing the rapid construction of highly bionic organs. This three-dimensional bioprinting technology based on acoustic fluid mediation has the characteristics of rapidity and reusability, as well as the advantages of adjustable cell concentration and no need for any modification of the cells.

[0018] Specifically, the present invention generates a vortex through an array-focused surface acoustic wave and a circular sound field boundary. First, each focusing-type interdigital electrode generates a focused pulsed surface acoustic wave on the surface of the piezoelectric lithium niobate substrate. Then, the propagation coupling enters the flat cylindrical liquid layer. Due to the attenuation effect of the sound wave during the propagation process, the directional flow of the liquid is caused in the propagation direction of the surface acoustic wave, which is manifested in the form of a liquid pulse. Then, since the liquid pulse does not point to the center of the circular sound field boundary, it forms a vortex liquid flow under the constraint of the circular sound field boundary. This liquid vortex flow effect is simultaneously enhanced by multiple focusing-type interdigital electrodes. Finally, driven by this vortex, the cells in the liquid will be enriched in the central area within the circular sound field boundary, thereby achieving precise and controllable enhancement of cell density.

[0019] The present invention is a cell manipulation method based on acoustic fluid, which can perform remote and contactless manipulation on cells to achieve rapid increase in cell density. At the same time, the printing accuracy can reach 10μm level, and centimeter-level tissue structures can be quickly printed within half an hour, completing tissue engineering construction with a cell density of one billion per milliliter. The present invention can construct highly bionic tissues and has great functional improvements. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It is a schematic diagram of the structure of a sedimentation-type bioprinting device for ultra-high cell density functional organs mediated by acoustic fluid; wherein A is a schematic diagram of the structure of an array surface acoustic wave chip; and B is a schematic diagram of the structure of the bioprinting device;

[0021] Figure 2 is a schematic diagram of focusing the surface acoustic wave field to gather cells;

[0022] Figure 3It is to prepare ultra-high-density three-dimensional human-shaped cell structure real map; a~e correspond to the foot, knee, waist and human cell structure respectively;

[0023] Figure 4 It is a picture of the actual preparation of ultra-high-density liver and alveolar tissue and its functional characterization results; A is the bioprinted vascularized liver tissue, and Ⅱ in the picture is the blood flow; B is the molecular level verification of the enhanced function of bionic liver tissue, ALB and TAT in the left picture are the albumin gene and tyrosine transaminase of liver parenchymal cells, and ALB in the right picture is albumin; C is the reproduction of alveolar respiratory function. DETAILED DESCRIPTION

[0024] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments.

[0025] like Figure 1 As shown, the present invention provides an acoustic fluid-mediated sedimentation bioprinting device for ultra-high cell density functional organs, including an array surface acoustic wave chip 1, a printing container 2, a printing platform 3, a curing light source 4 and a print head 5.

[0026] The printing container 2 is used to contain printing ink, which is a mixture of cells and photocurable biological ink.

[0027] The printing platform 3 is horizontally arranged in the printing container 2 and can move up and down in the printing ink.

[0028] The print head 5 is fixedly located above the print container 2 . The array surface acoustic wave chip 1 is fixed at the lower end of the print head 5 .

[0029] The array surface acoustic wave chip 1 includes a plurality of interdigital electrodes 11 arranged in a circular ring in the same horizontal plane. The interdigital electrodes 11 are focusing surface acoustic wave interdigital electrodes. The central axis of each interdigital electrode 11 deviates from the direction toward the center of the circle (i.e., deviates from the line connecting it and the center of the circle), and the deviation directions of the plurality of interdigital electrodes 11 are the same, all deviating clockwise or counterclockwise.

[0030] The array surface acoustic wave chip 1 is in contact with the printing ink (it can be in contact with the liquid surface of the printing ink or can be extended into the printing ink to achieve contact), and can form a focused acoustic flow in the printing ink between it and the printing platform 3 to gather cells in the printing ink.

[0031] The curing light source 4 is fixed in the print head 5 and is located above the array surface acoustic wave chip 1. The curing light source 4 can be projected into the printing ink above the printing platform 3 and cure it.

[0032] During printing, the transparent array surface acoustic wave chip 1 is used as the upper interface of sedimentation printing to level the liquid surface.

[0033] Preferably, in the array surface acoustic wave chip 1, the deflection angles of the multiple finger electrodes are the same, and the deviation angle is: 0°<deviation angle<90°, for example, 15°, 20°, etc. And the multiple finger electrodes are evenly distributed on the ring, for example, the spacing angle of 6 finger electrodes is 60°, and the spacing angle of 8 finger electrodes is 45°. The multiple finger electrodes 11 share the same piezoelectric substrate, that is, multiple electrode patterns are processed on one piezoelectric substrate. The number of electrode pairs in the finger electrode 11 is 3 to 10 pairs, for example, 5 pairs, 6 pairs, etc., and the wavelength is 200 to 350μm, for example, 270μm, 300μm, etc. The driving signal of the array surface acoustic wave chip 1 is a sinusoidal alternating current input with a voltage of 250mVpp.

[0034] The fabrication of the array surface acoustic wave chip is based on the semiconductor preparation process. First, the electrode arrangement is designed using computer-aided software, and then the metal electrodes are processed on the piezoelectric substrate using standard soft lithography and electron beam metal deposition technology. In a specific embodiment, the circular working area is designed to have a diameter of 1 cm; the wavelength, arrangement and orientation of the interdigitated electrodes are designed using AutoCAD software; the mask of the electrode is obtained by thermoplastic molding, and the AZ series positive photoresist is spin-coated on the lithium niobate piezoelectric substrate, and the electrode pattern on the mask is obtained by replication and elution; then a 5nm thick chromium film and a 50nm thick gold film are deposited on the piezoelectric substrate using electron beam evaporation; finally, the pre-designed array surface acoustic wave chip is eluted in formaldehyde at 60°C. The piezoelectric substrate is fixed to the print head, and the electrode pattern (gold electrode) faces the printing ink.

[0035] Preferably, the printing container 2 can also move up and down, so that thicker tissues and organs can be printed.

[0036] The present invention also provides a printing method of the acoustic fluid-mediated sedimentation-type biological printing device for ultra-high cell density functional organs.

[0037] The printing method of a single-layer tissue organ comprises the following steps:

[0038] S1.1. Expose a circular projection through a curing light source to solidify the printing ink to form a circular sound field boundary.

[0039] S1.2. Turn on the array surface acoustic wave chip to form a focused surface acoustic wave acoustic field, induce a fluid vortex within the boundary of the acoustic field, and thus gather the cells in the printed ink.

[0040] S1.3. Expose the projection of the shape to be printed through a curing light source, so that the printing ink is cured to form a tissue organ of the shape to be printed containing aggregated cells.

[0041] S1.4. Expose the projection containing the shape of the printed tissue and organ through a curing light source, so that the printing ink is cured to form a single-layer printed structure containing the tissue and organ of step three.

[0042] The printing method of multi-layer tissue organs comprises the following steps:

[0043] S1. First, print a single-layer printing structure according to the printing method of a single-layer tissue organ.

[0044] S2, move the printing platform 3 downward, repeat S1.1 to S1.4, and print a second layer of printing structure on top of the single layer of printing structure.

[0045] S3. Repeat S2 n times to obtain an n+2-layer printing structure, where n≥0.

[0046] Among them, the distance between the lower surface of the array surface acoustic wave chip 1 / previous layer of printed structure and the printing platform 3 is the thickness of the layer of printed structure. That is, in S1, the distance between the array surface acoustic wave chip 1 and the printing platform 3 is the thickness of the printed single layer of tissue. In S2, the distance between the lower surface of the previous layer of printed structure and the printing platform 3, that is, the distance that the printing platform 3 descends, is the thickness of the layer of printed structure.

[0047] The present invention achieves the phenomenon of cell aggregation in liquid by adjusting the distribution of the focused surface acoustic wave acoustic field array and the boundary conditions of the liquid. By adjusting the strength of the acoustic field, the thickness of the liquid layer and the initial cell concentration, different cell aggregation speeds, areas and degrees are obtained. Specifically, a plurality of interdigital electrodes deflected in the same direction and pointing to the center of the circle generate surface acoustic waves, and then a liquid vortex is formed by using the circular sound field boundary, that is, a stable focused acoustic flow is generated by the array surface acoustic wave chip, thereby achieving the enrichment of cells in the liquid toward the center, and specifically, the cells in the printed ink can be efficiently and controllably gathered in the central area at a layer thickness of 40μm. Increasing or decreasing the power intensity of the signal input of the array surface acoustic wave chip will speed up or slow down the focused acoustic flow, and the time and range of cell enrichment in the central area will increase and decrease accordingly. Changing the deflection angle of the interdigital electrodes in the array surface acoustic wave chip will also change the corresponding focused acoustic flow, thereby regulating the aggregation state of the cells.

[0048] The present invention uses an array of surface acoustic wave chips and integrates a sedimentation stereolithography process to achieve remotely controllable concentration and aggregation of cells in photo-crosslinkable biological ink in stereolithography, thereby achieving ultra-high-density three-dimensional bioprinting. For example, it can achieve density-adjustable spatial structure construction of hepatocytes, alveolar epithelial cells, and fibroblasts, and at the same time, a vascular structure can be realized inside.

[0049] In a specific embodiment, Figure 1As shown in Figure A, a 1cm diameter array surface acoustic wave chip containing 6 interdigitated electrodes with a center orientation of 15° counterclockwise deflection is produced using standard soft lithography technology in semiconductor processing technology. The interdigitated electrodes are designed to have 5 pairs of electrodes and a wavelength of 270μm. Figure 2 As shown, within the circular acoustic field boundary and driven by a sinusoidal AC signal with an amplitude voltage of 250mVpp, a liquid vortex I in the central area is presented and the cells are continuously sucked and dragged. Printing ink is added to the printing container: 10% (v / v) gelatin, photoinitiator ruthenium (Ru) / sodium persulfate (SPS) (1mM / 10mM) and cells with a cell density of one million per milliliter. The printing ink can also be: 10% (v / v) methacrylate hydrogel (GelMA), 1% photoinitiator phenyl-2,4,6-trimethylbenzoylphosphonic acid lithium (LAP) and cells with a cell density of one million per milliliter.

[0050] First, a circular hydrogel boundary with a diameter of 1 cm is formed by exposure (sound field boundary). Then, the focused surface acoustic wave field is stimulated. After 20 seconds of cell aggregation, cells in the central area form close contact. Then, the structure to be printed is projected, with an exposure time of 5 seconds and an exposure intensity of 50 mW / cm 2 Finally, the entire layer of printing ink containing cells is solidified to obtain a high-density, high-mechanical-strength two-dimensional cell gel sheet.

[0051] The programming software controls the printing platform to move down 40 μm to print the next layer of cells. After waiting for 15 seconds for the cells to rest, the steps of the first layer are repeated again: printing the acoustic field boundary, aggregating cells, patterning and polymerizing the printing ink, and curing the entire layer of printing ink. This cycle is repeated to finally obtain a complex tissue structure with high cell density, such as Figure 3 The height of the humanoid structure is 1.5 mm, and the layer thickness accuracy is 40 μm.

[0052] In another specific embodiment, the target organ cells, liver parenchymal cells, are used to print a high-density bionic liver structure layer by layer to achieve rapid maturation of liver tissue model functions, such as Figure 4 As shown in the figure, using hepatocytes, bionic liver tissue is realized, which has a bionic structure and reproduces the vascular structure. The close cell contact enables the expression of specific functional genes of hepatocytes and the enhancement of the secretory function of liver tissue.

[0053] In another specific embodiment, the target organ cells, alveolar epithelial cells, are used to print high-density bionic alveolar structures layer by layer, and the respiratory function of the alveolar tissue model is reproduced, such as Figure 4 shown.

[0054] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the reagents, materials and operating procedures used herein are reagents, materials and conventional procedures widely used in the corresponding fields.

[0055] It should be noted that the terms such as "upper", "lower", "left", "right", "front", "back", etc. cited in the invention are only for the convenience of description and are not used to limit the scope of implementation of the present invention. Changes or adjustments in their relative relationships should be regarded as the scope of implementation of the present invention without substantially changing the technical content.

[0056] Finally, it should be noted that the above are only preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. 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. An acoustic fluid-mediated sedimentation bioprinting device for ultra-high cell density functional organs, characterized in that: It includes an array surface acoustic wave chip, a printing container, a printing platform and a curing light source; The printing container is used to hold printing ink, which is a mixture of cells and photocurable biological ink; The printing platform is horizontally arranged in the printing container and can move up and down in the printing ink; The array surface acoustic wave chip and the curing light source are both located above the printing container; The array surface acoustic wave chip comprises a plurality of interdigital electrodes arranged in a circular shape in the same horizontal plane; the interdigital electrodes are focusing surface acoustic wave interdigital electrodes; the central axis of each interdigital electrode deviates from the direction toward the center of the circle, and the deviation directions of the plurality of interdigital electrodes are the same; the plurality of interdigital electrodes share the same piezoelectric substrate; The array surface acoustic wave chip is in contact with the printing ink and is capable of forming a focused acoustic flow in the printing ink between the array surface acoustic wave chip and the printing platform to gather cells in the printing ink; The curing light source is projected into the printing ink above the printing platform and cures it.

2. The acoustic fluid-mediated sedimentation bioprinting device for ultra-high cell density functional organs according to claim 1, characterized in that: in, In the array surface acoustic wave chip, the deflection angles of the plurality of interdigital electrodes are the same.

3. The acoustic fluid-mediated sedimentation bioprinting device for ultra-high cell density functional organs according to claim 1, characterized in that: in, The deviation angle of the interdigital electrodes is: 0°<deviation angle<90°.

4. The acoustic fluid-mediated sedimentation bioprinting device for ultra-high cell density functional organs according to claim 1, characterized in that: in, In the array surface acoustic wave chip, a plurality of interdigital electrodes are evenly distributed.

5. The acoustic fluid-mediated sedimentation bioprinting device for ultra-high cell density functional organs according to claim 1, characterized in that: in, The number of electrode pairs in the interdigitated electrode is 3 to 10 pairs, and the wavelength is 200 to 350 μm.

6. The acoustic fluid-mediated sedimentation bioprinting device for ultra-high cell density functional organs according to claim 1, characterized in that: in, The printing container can move up and down.

7. The acoustic fluid-mediated sedimentation bioprinting device for ultra-high cell density functional organs according to claim 1, characterized in that: in, The device also includes a printing head; the array surface acoustic wave chip is fixed at the bottom of the printing head.

8. The printing method of the sedimentation bioprinting device for ultra-high cell density functional organs mediated by acoustic fluid as described in any one of claims 1 to 7, characterized in that: The printing method of a single-layer tissue organ comprises the following steps: S1.1, exposing the printed ink to the curing light source to form a circular sound field boundary; S1.2, turning on the array surface acoustic wave chip to form a focused surface acoustic wave acoustic field, inducing a fluid vortex within the boundary of the acoustic field, thereby gathering cells in the printing ink; S1.3, exposing the ink to the curing light source to solidify the printing ink into a tissue organ of a to-be-printed shape containing aggregated cells; S1.

4. Expose the printed ink through the curing light source to cure the printed ink into a single-layer printed structure containing the tissue and organ of step three.

9. The printing method of the sedimentation-type bioprinting device for ultra-high cell density functional organs mediated by acoustic fluid according to claim 8, characterized in that: The printing method of multi-layer tissue organs comprises the following steps: S1, first printing a single-layer printed structure according to the printing method of the single-layer tissue organ; S2, move the printing platform downward, repeat S1.1 to S1.4, and print the second layer of printing structure on top of the single layer of printing structure; S3. Repeat S2 n times to obtain an n+2-layer printing structure, where n≥0.

10. The printing method of the sedimentation-type bioprinting device for ultra-high cell density functional organs mediated by acoustic fluid according to claim 9, characterized in that: in, In S1, the distance between the array surface acoustic wave chip and the printing platform is the thickness of the printed single layer of tissue; in S2, the distance between the lower surface of the previous layer of printed structure and the printing platform is the thickness of the layer of printed structure.

Images