Oblique-incident-body-wave-assisted ultra-high-cell-density multicellular tissue lifting bioprinting device and printing method therefor

Abstract

The present invention relates to the technical field of medical manufacturing. Disclosed are an oblique-incident-body-wave- assisted ultra-high-cell-density multicellular tissue lifting bioprinting device and a printing method therefor, the bioprinting device comprising a printing liquid tank, a printing platform, a light source and an oblique incident body wave chip, wherein the printing liquid tank contains a printing liquid which is a mixture of cells and a photocurable bio-ink; the printing platform is arranged horizontally and extends into the printing liquid tank to come into contact with the printing liquid; the printing platform can be raised or lowered; the light source is arranged below the printing liquid tank, and light is incident into the printing liquid tank; the oblique incident body wave chip is a piezoelectric transducer arranged obliquely; and the piezoelectric transducer is arranged below the printing liquid tank and tilted relative to a horizontal plane so as to generate an oblique incident body wave acoustic field in the printing liquid. The present invention can realize large-volume three-dimensional printing of a biomimetic tissue model with an ultra-high-cell-density and multicellular structure, and has the advantages of being ultra fast, having a large volume, and having an adjustable cell concentration and a simple liquid-exchange structure.

Description

An oblique-incidence body wave-assisted ultra-high cell density multicellular tissue pull-up bioprinting device and printing method thereof Technical Field

[0001] The present invention belongs to the field of medical manufacturing technology, and relates to a biological printing device, in particular to an oblique-incident body wave-assisted ultra-high cell density multicellular tissue pulling-type biological printing device and a printing method thereof. Background Art

[0002] Solid organs play a vital role in maintaining human physiological homeostasis, and the specific functions of these organs rely on specialized developmental processes that form close cellular biological contacts at the nanometer and micrometer levels. The biomimetic reconstruction of organs is a crucial research area with significant practical significance. Various technologies have been developed to reconstruct organs and tissues, aiming to reproduce their functional characteristics. Among them, 3D bioprinting utilizes bio-inks, cells, and additive manufacturing to create biomimetic living systems. Compared with other organ engineering technologies, it offers unique advantages such as high precision, multidimensionality, high degrees of freedom, high throughput, and integration.

[0003] Extrusion-based or pulling-based jetting and photolithography-based technologies, which polymerize cells through light, heat, or ultrasound, are the main strategies for 3D bioprinting. Jet-based bioprinting allows for the controllable deposition of cell-laden bioinks in the form of droplets and fibers through a nozzle or printhead, enabling the bottom-up engineering of living structures with precise cell types and arrangements. Photolithography-based bioprinting generates more complex 3D biostructures by projecting an image onto a photosensitive biomaterial, inducing layer-by-layer crosslinking. This approach enables precise and flexible control of tissue size and shape, providing ultrahigh resolution. However, these traditional printing methods are unable to achieve ultrahigh cell densities compared to solid native tissues (which typically have cell densities greater than 40 million cells per milliliter). This is because mechanical extrusion or pulling of high-cell-density bioinks in jet-based bioprinting often leads to cell damage, while photolithography-based bioprinting lacks effective and controllable cell assembly strategies. These bottlenecks limit the application of traditional 3D bioprinting. Summary of the Invention

[0004] To address the problem of low cell density in traditional three-dimensional bioprinting, the present invention provides an oblique-incident body wave-assisted ultra-high cell density multicellular tissue pulling-type bioprinting device and its printing method, which can be used to produce biomimetic in vitro tissues with real organ density and multicellularity, and can be applied to drug screening, disease research, organ transplantation and other fields.

[0005] To achieve the above objectives, the present invention provides an oblique-incident body wave-assisted ultra-high cell density multicellular tissue pull-up bioprinting device, which has the following characteristics: it includes a printing liquid tank, a printing platform, a light source and an oblique-incident body wave chip; the printing liquid tank contains a printing liquid mixed with cells and a photocurable biological ink; the biological ink can be a photocurable reagent such as a hydrogel solution; the printing platform is arranged horizontally and extends into the printing liquid tank, contacting the printing liquid (it can contact the liquid surface of the printing liquid or extend into the printing liquid to achieve contact); the printing platform can rise and fall; the light source is arranged below the printing liquid tank, and light is incident into the printing liquid tank for curing the printing liquid; the oblique-incident body wave chip is an obliquely arranged piezoelectric transducer; the piezoelectric transducer is arranged below the printing liquid tank, inclined relative to the horizontal plane, pointing to the center of the printing liquid tank, and generates an oblique-incident body wave acoustic field in the printing liquid.

[0006] Furthermore, the present invention provides an oblique-incident body wave-assisted ultra-high cell density multicellular tissue pulling-type bioprinting device, which may also have the following characteristics: wherein the tilt angle of the piezoelectric transducer is: 0°<tilt angle<90°; preferably 45°.

[0007] Furthermore, the present invention provides an oblique-incident body wave-assisted ultra-high cell density multicellular tissue pulling-type bioprinting device, which may also have the following characteristics: wherein the piezoelectric transducer is a piezoelectric ceramic, positive and negative electrodes are welded on the same surface of the piezoelectric ceramic, and the driving signal is a sinusoidal alternating current input with a voltage of 200 mVpp.

[0008] Furthermore, the present invention provides an oblique-incident body wave-assisted ultra-high cell density multicellular tissue pulling-type bioprinting device, which may also have the following characteristics: wherein the operating frequency of the piezoelectric transducer is 1-10 MHZ, preferably 2.06 MHz.

[0009] Furthermore, the present invention provides an oblique-incident body wave-assisted ultra-high cell density multicellular tissue pulling-type bioprinting device, which may also have the following characteristics: wherein the piezoelectric transducer is arranged in a medium that can conduct sound waves.

[0010] Furthermore, the present invention provides an oblique-incident body wave-assisted ultra-high cell density multicellular tissue pulling-type bioprinting device, which may also have the following characteristics: wherein, the printing device also includes an acoustic wave medium container; the acoustic wave medium container is arranged below the printing liquid tank and contains the medium that conducts acoustic waves; the piezoelectric transducer is arranged in the acoustic wave medium container.

[0011] Furthermore, the present invention provides an oblique-incident body wave-assisted ultra-high cell density multicellular tissue lifting-type biological printing device, which may also have the following characteristics: wherein, the printing device also includes a lifting platform; the printing platform is fixed under the lifting platform, and the lifting platform drives the printing platform to rise and fall.

[0012] The present invention also provides a printing method of an ultra-high cell density multicellular tissue pulling-type bioprinting device assisted by oblique-incident body waves, which has the following characteristics: the printing method of a single-layer tissue includes the following steps: S1.1, exposing a circular projection through the light source to solidify the printing liquid to form a circular sound field boundary; S1.2, turning on the oblique-incident body wave chip to form an oblique-incident body wave sound field, inducing a fluid vortex within the sound field boundary, thereby gathering cells in the printing liquid; S1.3, exposing a projection of the shape to be printed through the light source, so that the printing liquid solidifies into a tissue of the shape to be printed containing aggregated cells; S1.4, exposing a projection of the shape containing the printed tissue through the light source, so that the printing liquid solidifies into a single-layer printing structure containing the tissue in step three.

[0013] Furthermore, the present invention provides a printing method for an ultra-high cell density multicellular tissue pulling-type bioprinting device assisted by oblique-incident body waves, which may also have the following characteristics: wherein, the printing method for multi-layer tissue is: S1, first print a single-layer printing structure according to the printing method for single-layer tissue; S2, lift the printing platform, repeat S1.1-S1.4, and print a second-layer printing structure under the single-layer printing structure; S3, repeat S2 n times to obtain an n+2-layer printing structure, n≥0.

[0014] Furthermore, the present invention provides a printing method for an ultra-high cell density multicellular tissue pulling-type bioprinting device assisted by oblique-incident body waves, which may also have the following characteristics: wherein, the distance between the printing platform and the bottom surface of the printing liquid tank is the thickness of the printing structure layer.

[0015] The beneficial effects of the present invention are as follows: the present invention discloses an oblique-incident body wave-assisted ultra-high cell density multicellular tissue pull-up bio-printing device and a printing method thereof, which can perform large-volume three-dimensional printing of bionic tissue models with ultra-high cell density and multicellular structure. The present invention is a cell manipulation method based on body acoustic wave fluid, which can perform remote and contactless manipulation of cells to achieve rapid increase in cell density. Specifically, the present invention gathers cells on the central area of ​​pre-polymerization based on the large-volume eddy current caused by oblique-incident body waves, and obtains a cell hydrogel carrier with a cell density equivalent to that of natural tissue; then, through an integrated pull-up stereolithography process, a bionic cell structure with complex three-dimensional layers can be generated. The present invention can realize the architecture of any multi-cellular component by combining an acoustically assisted medium exchange system.

[0016] Specifically, the present invention generates vortices through obliquely incident body waves and a circular acoustic field boundary. First, the piezoelectric sensor is activated to generate a plane wave, which then propagates obliquely into the printing liquid tank. Due to the attenuation effect of the acoustic wave, this plane wave propagation causes a directional flow of the printing liquid within the printing liquid tank, manifesting as a liquid pulse. The liquid pulse then passes through the constraints of the circular acoustic field boundary to form a vortex-shaped liquid flow. Finally, driven by this vortex, the cells in the printing liquid are enriched in the central area of ​​the printing liquid tank, thereby achieving a significant dynamic increase in cell density.

[0017] The present invention significantly improves the cell aggregation flux induced by sound waves, increases the cell concentration in the hydrogel, and can also achieve the rapid construction of highly bionic multicellular organs. It has the advantages of ultra-fast speed, large volume, adjustable cell concentration and simple fluid replacement structure. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 is a schematic diagram of the structure of an oblique-incident body wave-assisted ultra-high cell density multicellular tissue pull-type bioprinting device, wherein A is a physical image of the oblique-incident body wave chip and a schematic diagram of the oblique-incident body wave acoustic field, and B is a schematic diagram of the structure of the printing device, wherein 1 is the oblique-incident body wave chip, 2 is the printing platform, 3 is the printing liquid tank, 4 is the light source, 5 is the lifting platform, 6 is the acoustic wave medium container, 7 is the printing liquid, 8 is the vortex acoustic flow caused by the body wave, and 9 is the incident acoustic wave;

[0019] Figure 2 is a two-dimensional cell aggregation diagram, where A is a sparse cell sheet without acoustic aggregation, and B is a high-density cell sheet with acoustic aggregation;

[0020] Figure 3 is a diagram of the multi-cell printing process, where A is the first cell printing and B is the second cell printing;

[0021] Figure 4 is a physical picture of the printed ultra-high-density multicellular tissue, where A is the three-dimensional vascular structure and B is the arteriovenous structure. DETAILED DESCRIPTION

[0022] The specific embodiments of the present invention are described below with reference to the accompanying drawings.

[0023] As shown in FIG1 , the present invention provides an oblique-incident body wave-assisted ultra-high cell density multicellular tissue pulling-type bioprinting device, comprising an oblique-incident body wave chip 1 , a printing platform 2 , a printing liquid tank 3 and a light source 4 .

[0024] The printing liquid tank 3 contains a printing liquid 7 , which is a mixture of cells and a photocurable biological ink. The biological ink can be a photocurable reagent such as a hydrogel solution.

[0025] The printing platform 2 is arranged horizontally and extends into the printing liquid tank 3 to contact the printing liquid. Specifically, the printing platform 2 can contact the liquid surface of the printing liquid or extend into the printing liquid to achieve contact.

[0026] The printing platform 2 can rise and fall. Specifically, the printing device further includes a lifting platform 5. The printing platform 2 is fixed below the lifting platform 5, and the lifting platform 5 drives the printing platform 2 to rise and fall.

[0027] The light source 4 is disposed below the printing liquid tank 3 , and light is incident into the printing liquid tank 3 to solidify the printing liquid.

[0028] The oblique-incident bulk wave chip 1 is an inclined piezoelectric transducer. The piezoelectric transducer is arranged below the printing liquid tank 3, tilted relative to the horizontal plane, pointing toward the center of the printing liquid tank 3, and generating an oblique-incident bulk wave acoustic field in the printing liquid. The tilt angle of the piezoelectric transducer is: 0°<tilt angle<90°, preferably 45°. The operating frequency of the piezoelectric transducer is 1-10 MHz, preferably 2.06 MHz. Specifically, the piezoelectric transducer is a piezoelectric ceramic. Positive and negative electrodes are welded on the same surface of the piezoelectric ceramic, and the driving signal is a sinusoidal AC input with a voltage of 200 mVpp.

[0029] In a preferred embodiment, the piezoelectric transducer is disposed in a medium capable of conducting sound waves. Specifically, the printing device further includes an acoustic medium container 6. This container, disposed below the printing liquid tank 3, contains the acoustic medium. The piezoelectric transducer is disposed within this container and can be secured using a rigid bracket printed by the 3D printer.

[0030] The present invention also provides a printing method of the oblique-incident body wave-assisted ultra-high cell density multicellular tissue pulling-type biological printing device.

[0031] The printing method of a single layer of tissue includes the following steps:

[0032] S1.1. Expose a circular projection through a light source to solidify the printing liquid to form a circular sound field boundary.

[0033] S1.2. Turn on the oblique-incident bulk wave chip to form an oblique-incident bulk wave acoustic field, which induces a fluid vortex within the acoustic field boundary, thereby gathering cells in the printing fluid.

[0034] S1.3. Expose a projection of the shape to be printed using a light source, so that the printing liquid solidifies into a tissue of the shape to be printed containing aggregated cells.

[0035] S1.4. Expose a projection of the shape of the printed tissue using a light source, causing the printing liquid to solidify into a single-layer printed structure containing the tissue from step 3.

[0036] The printing method of multi-layer tissue is:

[0037] S1. First, print a single-layer printing structure according to the printing method of a single-layer structure;

[0038] S2. Lift the printing platform and repeat S1.1-S1.4 to print a second layer of printing structure below the single-layer printing structure.

[0039] S3. Repeat S2 n times to obtain an n+2-layer printed structure, where n≥0.

[0040] The distance between the printing platform and the bottom of the printing tank is the thickness of the printed structure. That is, in S1, the distance between the printing platform and the bottom of the printing tank is the thickness of the printed single layer of tissue; in S2, the distance the printing platform is raised is the thickness of the printed structure.

[0041] The present invention generates body waves through an obliquely set piezoelectric transducer, and then uses a circular boundary to form a liquid vortex; by adjusting the spatial position of the oblique incident body wave acoustic field and the liquid boundary conditions, a large-scale cell aggregation phenomenon in the liquid is achieved; by adjusting the sound field strength, the liquid layer thickness, and the initial cell concentration, different cell aggregation speeds, areas, and degrees are obtained. Specifically, the oblique incident body wave chip can generate a stable focused acoustic flow, thereby achieving the enrichment of cells in the liquid toward the center. Increasing or decreasing the power intensity of the signal input of the oblique incident body 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 pointing deflection angle of the piezoelectric transducer of the oblique incident body wave chip will also change the corresponding focused acoustic flow, thereby regulating the aggregation state of the cells. The design and preparation method of the oblique incident body wave chip is: first, the finite element analysis simulation computer software COMSOL is used to design and optimize the acoustic flow induced by the piezoelectric transducer in the printing liquid tank, and then the effect is verified by means of controlled variable experiments. The oblique-incident bulk wave chip is incident directly from the oblique bottom of the pull-type printer, which does not affect the propagation and projection of the projection light source.

[0042] By integrating an optimized oblique-incidence bulk wave chip with a pull-up stereolithography process, the remotely controlled concentration and aggregation of cells within the photocrosslinkable bio-ink within the stereolithography can be achieved, enabling ultra-high-density 3D bioprinting. For example, one or more cell types, such as endothelial cells, smooth muscle cells, and fibroblasts, can be constructed into spatial structures with adjustable density, while also enabling the creation of vascular structures within them. Printing areas can reach the centimeter level, significantly improving printing efficiency and speed.

[0043] In one specific embodiment, a 0.5 × 5 × 10 mm piezoelectric transducer operating at 2.06 MHz was placed beneath a pull-type printer, deflected 45° clockwise toward the center of a liquid tank, creating an oblique-incidence bulk wave chip. The printing tank was filled with a printing solution consisting of 10% (v / v) gelatin, a photoinitiator of ruthenium (Ru) / sodium persulfate (SPS) (1 mM / 10 mM), and cells at a density of 1 million cells per milliliter.

[0044] First, a 4 cm diameter circular hydrogel boundary (the circular acoustic field boundary) was formed by exposure. The acoustic field was then stimulated. Driven by a sinusoidal AC signal with a 200 mVpp amplitude voltage, a vortex of liquid formed within the 4 cm diameter circular hydrogel boundary, continuously attracting and pulling cells. After 40 seconds of cell aggregation, the cells in the central region formed close contact, as shown in Figure 2. The desired printed structure was then projected, with an exposure time of 5 seconds and an exposure intensity of 60 mW / cm². Finally, the entire layer of printed liquid containing cells was cured, resulting in a high-density, mechanically strong, two-dimensional cell gel sheet.

[0045] Programming software controls the printing platform to move down 40 µm to print the next layer of cells. After waiting 15 seconds for the cells to settle, the process for the first layer is repeated: printing the acoustic field boundary, acoustofluidically aggregating cells, patterning the bio-ink, and curing the entire bio-ink layer. This cycle repeats until a complex tissue structure with a high cell density is achieved.

[0046] The device may also be provided with a plurality of tilted piezoelectric transducers to adjust the rotation speed of the generated sound field vortex.

[0047] This device can also perform multi-cell printing, meaning different layers of cells are printed. Between printing layers, the piezoelectric transducer can be activated (without an acoustic field boundary) to allow the printing fluid to flow out of the printing fluid reservoir. Then, another printing fluid can be replaced to print the next layer, as shown in Figure 3.

[0048] Utilize cells of the target organ, such as vascular endothelial cells and smooth muscle cells, to print out high-density biomimetic multicellular structures including vascular tissue, etc., or use different vascular endothelial cells to print out high-density biomimetic multicellular structures including arteriovenous tissue, etc., as shown in Figure 4.

[0049] 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 those widely used in the corresponding fields.

[0050] 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 to their relative relationships should be regarded as the scope of implementation of the present invention without substantially changing the technical content.

[0051] Finally, it should be noted that the above are merely 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 will be able to modify the technical solutions described in the aforementioned embodiments or substitute equivalents for some of the technical features. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

[0052]

Claims

1. An oblique-incidence body wave-assisted ultra-high cell density multicellular tissue pull-up bioprinting device, characterized by: The device comprises a printing liquid tank, a printing platform, a light source and an oblique-incidence body wave chip; the printing liquid tank contains a printing liquid mixed with cells and photocurable biological ink; The printing platform is arranged horizontally and extends into the printing liquid tank to contact with the printing liquid; The printing platform can rise and fall; the light source is set below the printing liquid tank, and the light is incident on the printing liquid tank; the oblique-incident body wave chip is an inclined piezoelectric transducer; the piezoelectric transducer is set below the printing liquid tank and is inclined relative to the horizontal plane, generating an oblique-incident body wave acoustic field in the printing liquid.

2. The oblique-incidence body wave-assisted ultra-high cell density multicellular tissue pull-up bioprinting device according to claim 1, characterized in that: in, The tilt angle of the piezoelectric transducer is: 0°<tilt angle<90°.

3. The oblique-incidence body wave-assisted ultra-high cell density multicellular tissue pull-up bioprinting device according to claim 1, characterized in that: in, The piezoelectric transducer is piezoelectric ceramic.

4. The oblique-incidence body wave-assisted ultra-high cell density multicellular tissue pull-up bioprinting device according to claim 1, characterized in that: in, The operating frequency of the piezoelectric transducer is 1-10 MHZ.

5. The oblique-incidence body wave-assisted ultra-high cell density multicellular tissue pull-up bioprinting device according to claim 1, characterized in that: in, The piezoelectric transducer is arranged in a medium that can conduct sound waves.

6. The oblique-incidence body wave-assisted ultra-high cell density multicellular tissue pull-up bioprinting device according to claim 5, characterized in that: The printing device also includes an acoustic wave medium container; The sound wave medium container is arranged below the printing liquid tank and contains the medium for conducting sound waves; the piezoelectric transducer is arranged in the sound wave medium container.

7. The oblique-incidence body wave-assisted ultra-high cell density multicellular tissue pull-up bioprinting device according to claim 1, characterized in that: in, The printing device also includes a lifting platform; the printing platform is fixed below the lifting platform, and the lifting platform drives the printing platform to rise and fall.

8. The printing method of the oblique-incidence body wave-assisted ultra-high cell density multicellular tissue pull-up bioprinting device according to any one of claims 1 to 7, characterized in that: The printing method of a single layer of tissue includes the following steps: S1.

1. Exposing the printing liquid to the light source to form a circular sound field boundary; S1.

2. Turning on the oblique-incidence bulk wave chip to form an oblique-incidence bulk wave acoustic field, inducing a fluid vortex within the acoustic field boundary, thereby gathering cells in the printing fluid; S1.3, exposing the printing liquid to the light source to solidify the printed tissue into a shape containing aggregated cells; S1.

4. Expose the printing liquid through the light source to solidify it into a single-layer printing structure containing the tissue in step 3.

9. The printing method of the oblique-incidence body wave-assisted ultra-high cell density multicellular tissue pull-type bioprinting device according to claim 8, characterized in that: in, The printing method of multi-layer tissue is: S1, first print a single-layer printed structure according to the printing method of the single-layer structure; S2. Lift the printing platform and repeat S1.1-S1.4 to print a second layer of printing structure below the single-layer printing structure. S3. Repeat S2 n times to obtain an n+2-layer printed structure, where n≥0.

10. The printing method of the oblique-incidence body wave-assisted ultra-high cell density multicellular tissue pulling-type bioprinting device according to claim 8, characterized in that: in, The distance between the printing platform and the bottom surface of the printing liquid tank is the thickness of the printing structure of this layer.

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