An oblique-incident body wave-assisted ultra-high cell density multicellular tissue lifting bioprinting device and printing method thereof

Abstract

The present invention discloses an oblique-incident body wave-assisted ultra-high cell density multicellular tissue lifting-type biological printing device and a printing method thereof, which belongs to the field of medical engineering manufacturing technology. The printing device 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 photocurable biological ink; the printing platform is horizontally arranged and extends into the printing liquid tank to contact the printing liquid; the printing platform can rise and fall; the light source is arranged below the printing liquid tank, and the light is incident into the printing liquid tank; the oblique-incident body wave chip is an obliquely arranged piezoelectric transducer; the piezoelectric transducer is arranged below the printing liquid tank, and is inclined relative to the horizontal plane, and an oblique-incident body wave acoustic field is generated in the printing liquid. The present invention can three-dimensionally print a bionic tissue model with ultra-high cell density and multicellular structure in large volume, and has the advantages of ultra-fast, large volume, adjustable cell concentration and simple liquid replacement structure.

Description

Technical Field

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

[0002] Solid organs play a vital role in maintaining the physiological stability of the human body, and the specific functions of these body organs rely on the close biological contact of nanometers and micrometers formed by special development. The bionic reconstruction of organs is an important research field with great practical significance. Various technologies have been developed to reconstruct organs and tissues, aiming to reproduce their functional characteristics. Among them, 3D bioprinting uses bio-ink, cells and additive manufacturing to manufacture bionic life systems. Compared with other organ engineering technologies, it has the unique advantages of high precision, multi-dimensionality, high degree of freedom, high throughput and integration.

[0003] Extrusion-type or traction-type jetting technology and photolithography technology through light, heat or ultrasound polymerization are the main strategies for 3D bioprinting. Jet-based bioprinting can controllably deposit cell-filled bioinks in the form of droplets and fibers through a nozzle or print head, allowing bottom-up engineering of living structures with precise cell types and arrangements. Lithography-based bioprinting can generate more complex 3D biological structures by projecting images onto photosensitive biomaterials, inducing layer-by-layer cross-linking of materials. This method can precisely and flexibly control tissue size and shape, providing ultra-high layer resolution. Compared with solid natural tissues (cell density is generally greater than 40 million cells per milliliter), these traditional printing methods cannot achieve ultra-high cell density. Because in jet-based bioprinting, mechanical extrusion or traction of high-cell-density bioinks usually leads to cell damage, and there is a lack of effective and controllable cell assembly strategies in lithography-based bioprinting, these bottlenecks limit the application of traditional 3D bioprinting. Summary of the invention

[0004] In response to the problem of low cell density in traditional three-dimensional bio-printing, the present invention provides an oblique-incident body wave-assisted ultra-high cell density multicellular tissue pulling-type bio-printing device and a printing method thereof, which can be used to produce bionic 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-mentioned purpose, the present invention provides an oblique-incident body wave-assisted ultra-high cell density multicellular tissue lifting-type 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 horizontally arranged and extends into the printing liquid tank to contact with the printing liquid (it can contact with 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 the 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, is inclined relative to the horizontal plane, points 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 lifting-type bioprinting device, which may also have the following characteristics: wherein the inclination angle of the piezoelectric transducer is: 0°<inclination 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, and 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 200mVpp.

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

[0009] Furthermore, the present invention provides an oblique-incident body wave-assisted ultra-high cell density multicellular tissue lifting-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 bio-printing 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 multi-cellular tissue lifting-type biological printing device assisted by an oblique-incident body wave, 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 a shape to be printed through the light source, so that the printing liquid is solidified into a tissue of the shape to be printed containing aggregated cells; S1.4, exposing a projection of a shape containing a printed tissue through the light source, so that the printing liquid is solidified into a single-layer printing structure containing the tissue of step three.

[0013] Furthermore, the present invention provides a printing method for an ultra-high cell density multi-cellular tissue lifting-type bio-printing device assisted by oblique-incident body waves, which may also have the following characteristics: wherein, the printing method for multi-layer tissues is: S1, first printing a single-layer printing structure according to the printing method for single-layer tissues; S2, lifting the printing platform, repeating S1.1 to S1.4, and printing a second-layer printing structure under the single-layer printing structure; S3, repeating 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 lifting-type bio-printing 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 multi-cellular 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 multi-cellular structures. The present invention is a cell manipulation method based on body acoustic wave fluid, which can perform remote, contactless manipulation of cells to achieve rapid increase in cell density. Specifically, the present invention gathers cells in the central area of ​​pre-polymerization based on the large-volume eddy current caused by the oblique-incident body wave, 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 three-dimensional complex layers can be generated. The present invention can realize the architecture of any multi-cellular components by combining an acoustically assisted medium exchange system.

[0016] Specifically, the present invention generates a vortex through oblique incident body waves and circular sound field boundaries. First, the piezoelectric sensor is activated to generate a plane wave, which then propagates into the printing liquid tank at an oblique incidence. Due to the attenuation effect of the sound wave, the plane wave propagation will cause a directional flow of the printing liquid in the printing liquid tank, which is manifested in the form of a liquid pulse. Then, the liquid pulse is constrained by the circular sound field boundary to form a vortex-shaped liquid flow. Finally, driven by this vortex, the cells in the printing liquid will be enriched in the central area of ​​the printing liquid tank, thereby achieving a significant dynamic enhancement of the 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 rapid construction of highly bionic multicellular organs. It has the advantages of ultra-fast, large volume, adjustable cell concentration and simple liquid replacement structure. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 1 is a schematic diagram of the structure of an ultra-high cell density multicellular tissue lifting bioprinting device assisted by oblique-incident body waves, wherein A is a physical picture of an oblique-incident body wave chip and a schematic diagram of forming an oblique-incident body wave acoustic field, B is a schematic diagram of the structure of the printing device, 1 is an oblique-incident body wave chip, 2 is a printing platform, 3 is a printing liquid tank, 4 is a light source, 5 is a lifting platform, 6 is an acoustic wave medium container, 7 is a printing liquid, 8 is a vortex acoustic flow caused by the body wave, and 9 is an 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 This is a printed ultra-high-density multi-cellular tissue physical image, where A is the three-dimensional vascular structure and B is the arteriovenous structure. DETAILED DESCRIPTION

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

[0023] like Figure 1 As shown, the present invention provides an oblique-incident body wave-assisted ultra-high cell density multicellular tissue lifting-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 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 can 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 body wave chip 1 is an obliquely arranged piezoelectric transducer. The piezoelectric transducer is arranged below the printing liquid tank 3, is inclined relative to the horizontal plane, points to the center of the printing liquid tank 3, and generates an oblique-incident body wave acoustic field in the printing liquid. The inclination angle of the piezoelectric transducer is: 0°<inclination angle<90°, preferably 45°. The operating frequency of the piezoelectric transducer is 1~10MHZ, preferably 2.06MHz. 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 alternating current input with a voltage of 200mVpp.

[0029] In a preferred embodiment, the piezoelectric transducer is disposed in a medium that can conduct sound waves. Specifically, the printing device further includes a sound wave medium container 6. The sound wave medium container 6 is disposed below the printing liquid tank 3 and contains a medium that conducts sound waves. The piezoelectric transducer is disposed in the sound wave medium container 6, and its placement position can be fixed by a hard bracket printed by a three-dimensional printer.

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

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

[0032] S1.1. A circular projection is exposed by a light source to solidify the printing liquid to form a circular sound field boundary.

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

[0034] S1.3. Expose the projection of the shape to be printed through 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 the projection of the shape of the printed tissue through a light source, so that the printing liquid solidifies into a single-layer printed structure containing the tissue of step three.

[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, repeat S1.1 to S1.4, and print the second layer of printing structure below the single layer of 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 surface of the printing tank is the thickness of the printed structure. That is, in S1, the distance between the printing platform and the bottom surface of the printing tank is the thickness of the printed single layer of tissue; in S2, the distance the printing platform is lifted is the thickness of the printed structure.

[0041] The present invention generates body waves through an inclined 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 range of cell aggregation phenomena in the liquid is achieved; 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, 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 the control variable experimental means. The oblique incident body wave chip is directly incident from the bottom of the pull-type printer, which does not affect the propagation and projection of the projection light source.

[0042] Through the optimized oblique-incidence body wave chip and the integrated pull-up stereolithography process, the cells in the photo-crosslinkable biological ink in the stereolithography can be remotely and controllably concentrated and aggregated, thereby achieving ultra-high-density three-dimensional bioprinting, for example, density-adjustable spatial structures can be constructed for one or more of vascular endothelial cells, smooth muscle cells, and fibroblasts, while vascular structures can be realized inside. The printing area reaches the centimeter level, greatly improving the printing efficiency and speed.

[0043] In a specific embodiment, a piezoelectric transducer of 0.5×5×10 mm and operating frequency of 2.06 MHz is placed under the pull-type printer, with a clockwise deflection angle of 45° pointing to the center of the liquid tank, forming an oblique incidence bulk wave chip. The printing liquid tank is added with printing liquid: 10% (v / v) gelatin, photoinitiator ruthenium (Ru) / sodium persulfate (SPS) (1mM / 10mM) and cells with a cell density of one million per ml.

[0044] First, a 4 cm diameter circular hydrogel boundary (circular acoustic field boundary) was formed by exposure. Then the acoustic field was stimulated. Under the drive of a 200 mVpp amplitude voltage sinusoidal AC signal within the 4 cm diameter circular hydrogel boundary, a liquid vortex in the central area appeared and continuously sucked the cells. After 40 seconds of cell aggregation, the cells in the central area formed close contact. The cells before and after aggregation were as follows Figure 2 As shown. Then project the structure to be printed, the exposure time is 5 seconds, and the exposure intensity is 60mW / cm 2 Finally, the entire layer of printing liquid containing cells is solidified to obtain a high-density, high-mechanical-strength two-dimensional cell gel sheet.

[0045] The programming software controls the printing platform to move down 40μm 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, acoustofluid aggregation of cells, patterning polymerized bio-ink, and solidifying the entire layer of bio-ink. This cycle is repeated to finally obtain a complex tissue structure with high cell density.

[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] The device can also perform multi-cell printing, that is, cells in different layers of structure are different. When printing, between printing different layers of structure, the piezoelectric transducer can be turned on (there is no sound field boundary at this time) to make the printing liquid flow out of the printing liquid tank, and then another printing liquid is replaced to print the next layer, such as Figure 3 shown.

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

[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 reagents, materials and conventional procedures 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 in 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 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 oblique-incidence body wave-assisted ultra-high cell density multicellular tissue lifting bioprinting device, characterized in that: 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 photocurable biological ink; The printing platform is horizontally arranged and extends into the printing liquid tank to contact with the printing liquid; The printing platform can be raised and lowered; The light source is arranged below the printing liquid tank, and the light is incident into the printing liquid tank; The oblique-incident body wave chip is an obliquely arranged piezoelectric transducer; the piezoelectric transducer is arranged below the printing liquid tank, in a medium capable of conducting sound waves, and is inclined relative to the horizontal plane to generate an oblique-incident body wave acoustic field in the printing liquid.

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

3. The oblique-incidence body wave-assisted ultra-high cell density multicellular tissue lifting 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 lifting 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 lifting bioprinting device according to claim 1, Features: in, 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.

6. The oblique-incidence body wave-assisted ultra-high cell density multicellular tissue lifting bioprinting device according to claim 1, characterized in that: in, 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.

7. The printing method of the oblique-incident body wave-assisted ultra-high cell density multicellular tissue lifting-type bioprinting device according to any one of claims 1 to 6, characterized in that: The printing method of a single-layer tissue comprises the following steps: S1.1, exposing the printing liquid by the light source to solidify to form a circular sound field boundary; S1.2, turning on the oblique-incident body wave chip to form an oblique-incident body wave acoustic field, inducing a fluid vortex within the acoustic field boundary, thereby gathering cells in the printing liquid; S1.3, exposing the printing liquid to the light source so as to solidify the tissue in the shape of the to-be-printed tissue containing aggregated cells; S1.4, exposing the printing liquid to light to solidify the printing liquid into a single-layer printing structure containing the S1.3 tissue.

8. The printing method of the oblique-incidence body wave-assisted ultra-high cell density multicellular tissue lifting bioprinting device according to claim 7, Features: in, The printing method of multi-layer tissue is: S1, first printing a single-layer printing structure according to the printing method of the single-layer structure; S2, lift the printing platform, repeat S1.1 to S1.4, and print the second layer of printing structure below the single layer of printing structure; S3. Repeat S2 n times to obtain an n+2-layer printed structure, where n≥0.

9. The printing method of the oblique-incidence body wave-assisted ultra-high cell density multicellular tissue lifting-type bioprinting device according to claim 7, 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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