Standing wave assembly device, assembly vessel, assembly method and application

Abstract

The invention provides a standing wave assembly device, an assembly vessel, an assembly method and application, the standing wave assembly device comprises an assembly table and a curing assembly, the assembly table is used for placing an assembly carrier, a vertically through light hole is formed in the assembly table, the light hole is used for exposing the bottom of the assembly carrier, and the curing assembly comprises a light source and a heat source. The curing assembly is located below the light hole and used for curing the pre-assembly liquid in the assembly carrier from the bottom. The curing assembly is arranged below the assembly table, so that the curing assembly can carry out light curing or heat curing on the pre-assembly liquid in the assembly carrier from the bottom, the pre-assembly liquid on the bottom layer can be selectively stabilized by controlling the curing process, and a structural layer with the thickness smaller than that of the pre-assembly liquid is formed; the structure layer with the thickness smaller than the minimum thickness needed for forming a stable standing wave field can be manufactured, and therefore the standing wave assembling device can be used for constructing a multi-layer structure with a low interval. The standing wave assembly device is used in the technical field of advanced manufacturing.

Description

Technical Field

[0001] The present application relates to the field of advanced manufacturing technology, and in particular to a standing wave assembly device, an assembly vessel, an assembly method and applications. Background Art

[0002] Tissue and organ manufacturing is defined as the engineering and construction of bionic tissue and organ structures in vitro based on tissue engineering principles and using advanced biomanufacturing technologies for the transplantation, repair and replacement of diseased tissues and organs in vivo, as well as the construction of in vitro physiological and pathological models.

[0003] At present, there are two main strategies for tissue and organ biomanufacturing: biological 3D printing and bioassembly. Bioassembly refers to the manufacture of bionic multi-level structures by automatically assembling pre-formed modular units (including cells or cell aggregates, cells and biomaterial complexes) according to a pre-set 2D or 3D model. Bioassembly can establish tissue structures without external scaffolds and is a manufacturing strategy that is closer to the natural physiological state.

[0004] Faraday waves were proposed by British physicist Michael Faraday in 1831 and are defined as nonlinear standing waves caused by the acceleration generated by the vertical oscillation of a liquid layer. The Faraday wave bioassembly method can achieve the assembly of modules in the field within a few seconds through the acoustic radiation force or fluid force generated by sound waves. It has the characteristics of non-contact, non-invasive, and efficient molding, which can minimize damage to cells and show unique technical advantages and huge application potential in the engineering construction of tissues and organs.

[0005] However, early Faraday wave bioassembly technology was limited to single-layer assembly. Although the assembly units (such as cell spheres, cell-loaded microspheres, etc.) themselves have a certain three-dimensional structure, there is still a large gap compared with the complex multi-layer, anisotropic three-dimensional structure of tissues and organs in the body, which greatly limits the application prospects of Faraday wave bioassembly in tissue and organ manufacturing. In 2022, Xiao et al. first proposed the Faraday wave multi-layer assembly method, which expanded the Faraday wave bioassembly technology from single-layer assembly to multi-layer assembly by stacking the assembly chambers in situ layer by layer, using the solidified upper layer as the base for the assembly of the next layer.

[0006] It should be noted that for tissue and organ manufacturing, reducing the interlayer spacing is more conducive to interlayer cell connection and communication, and is closer to the goal of biomimetic construction of complex structures of natural tissues. However, the existing Faraday wave multilayer assembly method has the problem of too far distance between different layer patterns. For example, in the related technology CN114603846B, the pattern thickness is 1.5mm.

[0007] This is because Faraday wave bio-assembly requires a certain thickness of liquid layer. When the height of the assembly chamber is too low (generally <0.8mm), due to the strong surface tension formed by the thin liquid surface, it is difficult to stimulate a stable Faraday standing wave field to drive the assembly unit to achieve patterning (Research on the Effect of Faraday Wave-Mediated Hydraulic Drag on Microparticle Patterning and Its Application in Biomanufacturing, Liu Nian, Wuhan University, 2020). Therefore, the thickness of each layer in the existing multi-layer assembly method is often greater than 0.8mm, resulting in a large distance between patterns in different layers. Summary of the invention

[0008] The present application aims to solve at least one of the technical problems existing in the prior art. To this end, the present application proposes a standing wave assembly device, an assembly vessel, an assembly method and an application. The standing wave assembly device is conducive to constructing a multi-layer structure with a low spacing.

[0009] The present application also proposes that it can be used as an assembly carrier of a standing wave assembly device to accommodate an assembly vessel with a multi-layer structure.

[0010] The present application also provides a method for assembling a multilayer structure.

[0011] The present application also proposes a multi-layer structure manufactured by an assembly method.

[0012] The present application also proposes the application of an assembly method.

[0013] According to the standing wave assembly device provided in the present application, it includes an assembly table and a curing component, the assembly table is used to place an assembly carrier, the assembly table is formed with a vertical through light hole, the light hole is used to expose the bottom of the assembly carrier, the curing component includes a light source and a heat source, the curing component is located below the light hole, and the curing component is used to cure the pre-assembly liquid in the assembly carrier from the bottom.

[0014] The standing wave assembly device provided by the present application has at least the following technical effects: by arranging a curing component under the assembly table, the curing component can photo-cure or thermally cure the pre-assembly liquid in the assembly carrier from the bottom, and by controlling the curing process, it is possible to selectively stabilize (completely or partially cure) the pre-assembly liquid in the bottom layer to form a structural layer with a thickness less than the thickness of the pre-assembly liquid, which is conducive to producing a structural layer with a thickness less than the minimum thickness required to form a stable standing wave field. Therefore, the standing wave assembly device is conducive to constructing a multi-layer structure with a low spacing.

[0015] According to some embodiments of the present application, the heat source includes a temperature variable film, the temperature variable film is arranged at the bottom of the assembly table, the temperature variable film covers the light-transmitting hole, the material of the temperature variable film is a transparent material, and the light source is located below the temperature variable film.

[0016] According to some embodiments of the present application, the standing wave assembly device includes an excitation component, the excitation component includes a support arm and a transducer, the assembly table is arranged on the support arm, the support arm is arranged on the transducer, and the transducer is used to generate vibration to generate standing waves in the pre-assembly liquid through the support arm.

[0017] According to some embodiments of the present application, the standing wave assembly device includes a buckle, which is movably disposed on the assembly table, and the buckle is used to press and fix the assembly carrier.

[0018] The assembly vessel provided in the present application includes a vessel body and a bottom cover, wherein the vessel body is formed with a vertically penetrating assembly chamber, the bottom cover is arranged on the vessel body, the bottom cover closes the bottom of the assembly chamber, and the material of the bottom cover is a transparent material.

[0019] The assembly vessel provided by the present application has at least the following technical effects: the assembly vessel can be used as an assembly carrier of a standing wave assembly device, and by providing a transparent bottom cover, the preassembly liquid in the assembly vessel can be photocured from the bottom to achieve selective stabilization of the preassembly liquid in the bottom layer, and by providing a bottom cover with better thermal conductivity, heat is mainly transmitted from the bottom cover to the preassembly liquid to achieve selective stabilization of the preassembly liquid in the bottom layer, which is conducive to producing a structural layer with a thickness less than the minimum thickness required to form a stable standing wave field. Therefore, the assembly vessel is conducive to constructing a multi-layer structure with a low spacing.

[0020] The assembly method provided in the present application includes: adding a pre-assembly liquid into an assembly chamber, wherein the pre-assembly liquid includes an assembly unit and a biological ink, wherein the biological ink has photosensitivity and / or temperature sensitivity; after the assembly unit settles to the bottom layer of the pre-assembly liquid, applying a standing wave to aggregate the assembly unit in a pattern; stabilizing the bottom layer of the biological ink from the bottom to fix the patterned assembly unit to obtain a first structural layer; replenishing the pre-assembly liquid, and according to the above process, manufacturing the next structural layer based on the first structural layer.

[0021] According to the assembly method provided in the present application, at least the following technical effects are achieved: by patterning the assembly units after they have settled to the bottom layer, and then combining this with a method of stabilization from the bottom, only a portion of the bio-ink located at the bottom layer is selectively stabilized, thereby forming a structural layer with patterned assembly units at the bottom layer of the pre-assembly liquid. This allows the thickness of the structural layer to be less than the height of the pre-assembly liquid in the assembly chamber during patterning, thereby reducing the spacing between assembly units located in adjacent structural layers, and constructing a multilayer structure with a low spacing.

[0022] According to some embodiments of the present application, replenishing the pre-assembly liquid includes removing the pre-assembly liquid located in an upper layer of the assembly chamber and adding new pre-assembly liquid into the assembly chamber.

[0023] According to some embodiments of the present application, after removing the pre-assembly liquid and before adding new pre-assembly liquid, the structural layer is completely cured.

[0024] According to some embodiments of the present application, when patterning the assembly unit, the height of the pre-assembly liquid is greater than or equal to the minimum thickness required to form a stable standing wave field, and the thickness of the structural layer is less than the minimum thickness required to form a stable standing wave field.

[0025] According to some embodiments of the present application, the height of the pre-assembly liquid is greater than or equal to 0.8 mm.

[0026] According to some embodiments of the present application, the thickness of the structural layer is less than 0.5 mm.

[0027] According to some embodiments of the present application, the pre-assembly liquid is irradiated from the bottom at a set time to control the thickness of the structural layer.

[0028] According to some embodiments of the present application, the pre-assembly liquid is heated from the bottom for a set time to control the thickness of the structural layer.

[0029] According to some embodiments of the present application, the assembly unit and the biological ink are first mixed to form the pre-assembly liquid, which is then added to the assembly chamber.

[0030] According to some embodiments of the present application, the pre-assembly liquid in the assembly chamber is left to stand, so that the assembly unit sinks to the bottom layer of the pre-assembly liquid.

[0031] According to some embodiments of the present application, the bio-ink is photosensitivity and contains a curable precursor and an initiator.

[0032] According to some embodiments of the present application, the curable precursor includes at least one of methacrylic anhydride polymer, N-isopropylacrylamide, N,N-diethylacrylamide, N-ethylmethacrylamide and poly (N-vinylisobutyramide).

[0033] According to some embodiments of the present application, in the bio-ink, the mass volume concentration of the curable precursor is 2% to 20%, preferably 5% to 15%, and more preferably 5% to 10%.

[0034] According to some embodiments of the present application, the initiator includes at least one of phenyl-2,4,6-trimethylbenzoyl lithium phosphite, 1-hydroxycyclohexyl phenyl ketone, 2-methyl-1-(4-methylthiophenyl)-2-morpholinyl-1-propanone, 4-methylbenzophenone and isopropylthioxanthone.

[0035] According to some embodiments of the present application, in the bio-ink, the mass volume concentration of the initiator is 0.05% to 1%, preferably 0.05% to 0.5%, more preferably 0.1% to 0.5%, and further preferably 0.2% to 0.3%.

[0036] According to some embodiments of the present application, the assembly unit includes at least one of a natural polymer material and a synthetic polymer material.

[0037] According to some embodiments of the present application, the natural polymer material is selected from chitosan, collagen, gelatin, cellulose, sodium alginate and corresponding cell-loaded microspheres and drug-loaded microspheres.

[0038] According to some embodiments of the present application, the assembly unit is insoluble in the bio-ink.

[0039] According to some embodiments of the present application, the assembly unit has a micron size, which is 10 to 500 microns, preferably 50 to 400 microns, and more preferably 100 to 300 microns.

[0040] According to some embodiments of the present application, the synthetic polymer material is selected from polylactic acid, polylactic acid-glycolic acid copolymer, polycaprolactone, polyhydroxyalkanoic acid esters and corresponding cell-loaded microspheres and drug-loaded microspheres.

[0041] According to some embodiments of the present application, the bio-ink further contains a solvent, and the solvent includes at least one of water, an aqueous solution, and an organic solvent. The aqueous solution can be selected according to actual needs, as long as it does not react with the assembly unit, does not dissolve the assembly unit, and does not have a side effect on the curing of the bio-ink, such as a phosphate solution, an acetic acid solution, a sodium chloride solution, a cell culture medium, etc. The organic solvent can be selected according to actual conditions, as long as it does not react with the assembly unit, does not dissolve the assembly unit, and does not have a side effect on the curing of the bio-ink, such as ethanol, methanol, ethylene glycol, glycerol, N,N-dimethylformamide, dichloromethane, tetrahydrofuran, etc.

[0042] According to some embodiments of the present application, the material of the assembly chamber is selected from at least one of polytetrafluoroethylene, glass, polymethyl methacrylate, polystyrene, polydimethylsiloxane, and cycloolefin polymer (COC / COP).

[0043] The application of the assembly method provided in the present application has the corresponding beneficial effects provided by the assembly method, which will not be elaborated here. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] The accompanying drawings are used to provide further understanding of the technical solution disclosed in the present application and constitute a part of the specification. Together with the embodiments disclosed in the present application, they are used to explain the technical solution of the present application and do not constitute a limitation on the technical solution disclosed in the present application.

[0045] Figure 1 It is a schematic diagram of the overall structure of the standing wave assembly device of an embodiment of the present application;

[0046] Figure 2 is a partial structural schematic diagram of a standing wave assembly device according to an embodiment of the present application, wherein an exploded diagram is used to illustrate the placement of an assembly vessel and a heat curing component on an assembly table;

[0047] Figure 3 is a schematic cross-sectional structural diagram of an assembly vessel according to an embodiment of the present application;

[0048] Figure 4 It is a schematic flow chart of the assembly method of an embodiment of the present application;

[0049] Figure 5 is the patterned multilayer structure prepared in Example 1 of the present application;

[0050] Figure 6 is a patterned multilayer structure prepared in Example 2 of the present application;

[0051] Figure 7 is the patterned multilayer structure prepared in Example 3 of the present application;

[0052] Figure 8 is a patterned multilayer structure prepared in Example 4 of the present application;

[0053] Fig. 9 It is a patterned multilayer structure prepared in Example 5 of the present application.

[0054] Reference numerals:

[0055] Assembling table 110, assembling carrier 120, vessel body 121, bottom cover 122, assembling chamber 123, buckle 130;

[0056] Light source 210, heat source 220;

[0057] Support arm 310, transducer 320;

[0058] Sample rack 410, test tube 420, pipette 430;

[0059] Robot 510. DETAILED DESCRIPTION

[0060] The embodiments of the present application are described in detail below. The raw materials used in the following embodiments, unless otherwise specified, can be obtained from conventional commercial sources; the processes used, unless otherwise specified, all adopt conventional processes in the art. Examples of the embodiments are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present application, and cannot be understood as limiting the present application.

[0061] In the description of the present application, it should be understood that descriptions involving orientation, such as up, down, front, back, left, right, etc., indicating orientations or positional relationships, are based on the orientations or positional relationships shown in the accompanying drawings, and are only for the convenience of describing the present application and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be understood as a limitation on the present application.

[0062] In the description of this application, "several" means one or more, "more" means more than two, "greater than", "less than", "exceed", etc. are understood to exclude the number itself, and "above", "below", "within", etc. are understood to include the number itself. If there is a description of "first" or "second", it is only used for the purpose of distinguishing technical features, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features or implicitly indicating the order of the indicated technical features.

[0063] In the related art, a multilayer structure with multilayer patterned assembly units is assembled using standing waves. A liquid layer of a certain thickness is required to stimulate a stable standing wave field, otherwise the patterning quality of the assembly unit will be affected. Therefore, in the multilayer structure, the distance between the assembly units of different layers is relatively far.

[0064] To this end, the present application provides an assembly method that can construct a multilayer structure with a small spacing. For tissue and organ manufacturing, reducing the interlayer spacing is more conducive to interlayer cell connection and communication, and is closer to the goal of biomimetic construction of complex structures of natural tissues.

[0065] In addition, the present application also provides a multilayer structure manufactured by the assembly method, and the application of the assembly method.

[0066] The assembly method can be applied to at least any of the following:

[0067] A1) constructing an engineered bone-cartilage interface structure;

[0068] A2) Constructing the skin layer structure;

[0069] A3) Building vascular structures;

[0070] A4) Construction of cartilage, skin or vascular disease models;

[0071] A5) Preparation of tissue repair materials.

[0072] Exemplarily, the assembly method may include the following steps:

[0073] Step S100: adding a pre-assembly liquid into the assembly chamber, wherein the pre-assembly liquid includes an assembly unit and biological ink, and the biological ink has photosensitivity and / or temperature sensitivity.

[0074] Bio-ink refers to an ink material that has good biocompatibility and can be cured into a shape. Generally speaking, according to the curing method, bio-inks include ion cross-linking type, thermosensitive type, photosensitive type and shear type. Photosensitive bio-ink has the photosensitivity required by this application and can be cured into a gel state under the action of light. Thermosensitive bio-ink has the temperature sensitivity required by this application and can be cured into a gel state after a temperature change (such as heating or cooling).

[0075] Step S200: After the assembly unit sinks to the bottom layer of the pre-assembly liquid, a standing wave is applied to pattern the assembly unit.

[0076] It can be understood that the potential well formed by the standing wave at this time enables the assembly unit to form a pattern on the bottom layer of the pre-assembly liquid. Optionally, the standing wave includes at least one of a linear standing wave and a nonlinear standing wave, and the nonlinear standing wave includes a Faraday wave. Optionally, the waveform of the standing wave includes at least one of a sine wave and a cosine wave.

[0077] Optionally, the frequency of the standing wave can be 20 to 200 Hz, 30 to 100 Hz, preferably 50 to 80 Hz. At different frequencies, patterns of different shapes can be formed. For example, applying a 50 Hz sine wave can assemble the assembly units into a double-ring concentric circle pattern, and applying an 80 Hz sine wave can assemble the assembly units into a petal-shaped pattern. In addition, the amplitude of the standing wave can be 0.5 to 5 V, preferably 1 to 3 V, and more preferably 1 to 2 V.

[0078] The applied standing wave can drive the pre-assembled liquid to vibrate vertically up and down or laterally, or to vibrate vertically and laterally crosswise and alternately.

[0079] It is understandable that the standing wave is emitted by a standing wave generating device. Exemplarily, the standing wave generating device includes a signal generator, a power amplifier, an exciter, an oscilloscope, an acceleration sensor and a dynamic data collector. The signal generator emits an electrical signal, the power amplifier amplifies the signal and transmits it to the exciter, the exciter converts the electrical signal into a mechanical signal, and drives the assembly units placed on the exciter to assemble into a pattern. The oscilloscope is used to detect the electrical signal amplified by the power amplifier, and the data can be directly displayed on the screen of the oscilloscope. The acceleration sensor is used to detect the mechanical signal emitted by the exciter, and the dynamic data collector transmits the signal collected by the acceleration sensor to the computer for display.

[0080] In order to improve the quality of pattern formation, optionally, the assembly unit and the biological ink can be evenly mixed first, and then the assembly unit can be settled. Since the assembly unit is evenly distributed in the pre-assembly liquid, the assembly unit can be basically evenly distributed in the bottom layer of the pre-assembly liquid after sedimentation. In this way, when patterning, it can be ensured that every area of ​​the pattern has an assembly unit, and shape defects are less likely to occur.

[0081] For example, the assembly unit and the bio-ink may be first mixed to form a pre-assembly solution, and then added to the assembly chamber. Mixing outside the assembly chamber can facilitate operation and also facilitate the preparation of sufficient and precisely proportioned pre-assembly solution in advance.

[0082] In addition, illustratively, the preassembly liquid in the assembly chamber can be allowed to settle to the bottom. The standing time can be flexibly determined according to the specific parameters of the preassembly liquid. illustratively, the preassembly liquid is allowed to stand in the assembly chamber for 5s to 150s, preferably 10s to 60s.

[0083] Step S300: Stabilize the bottom layer of bio-ink from the bottom (referring to the bottom of the assembly chamber) to fix the patterned assembly units to obtain the first structural layer.

[0084] It can be understood that for photosensitive bio-ink, stabilization of the bottom layer of bio-ink is achieved by irradiating it from the bottom, and for temperature-sensitive bio-ink, stabilization of the bottom layer of bio-ink is achieved by heating or cooling it from the bottom (depending on the specific material of the bio-ink).

[0085] Specifically, the curing of photosensitive bio-ink is affected by the intensity of light, so when irradiated from the bottom up, the pre-assembly liquid in the assembly chamber is gradually solidified. By selecting the appropriate irradiation time and irradiation intensity, only a part of the bio-ink in the bottom layer can be selectively transformed into a stable state (referring to the solidified or semi-solidified state with poor fluidity formed after the bio-ink is cross-linked), while the rest of the bio-ink in the upper layer remains in a flowable liquid state. The stabilized bio-ink fixes the assembly unit, thereby forming the first structural layer.

[0086] Optionally, the fixed assembly unit may be irradiated for a predetermined set time to control the thickness of the structural layer and obtain a structural layer with a thickness that meets requirements.

[0087] Specifically, the curing of the thermosensitive bio-ink is affected by the heat it receives. Taking the thermosensitive bio-ink that is cured by heat as an example, when heated from the bottom, the heat is gradually transferred from bottom to top, and the pre-assembly liquid in the assembly chamber is also gradually cured from bottom to top. By selecting the appropriate heating time and temperature, only a part of the bio-ink at the bottom layer can be selectively transformed into a stable state, while the rest of the bio-ink at the upper layer remains in a flowable liquid state. The stabilized bio-ink fixes the assembly unit, thereby forming the first structural layer.

[0088] Optionally, the assembly chamber can be configured such that the thermal conductivity of the bottom wall is higher than that of the side walls. Preferably, the side walls are poor thermal conductors or insulating materials, while the bottom wall is a high thermal conductivity material, thereby inhibiting heat transfer to the side walls and preventing the pre-assembly liquid from solidifying from the outside to the inside.

[0089] Step S400: replenish the pre-assembly liquid, and manufacture the next structural layer based on the first structural layer according to the above process (ie, step S200 and step S300).

[0090] After the assembly unit is fixed to the structural layer, the remaining pre-assembly liquid does not contain the assembly unit, or contains only a small amount of assembly unit. By adding new pre-assembly liquid, the assembly unit reaches the required amount of material, so as to manufacture the next structural layer.

[0091] It is understandable that when manufacturing the next structural layer, the new assembly unit settles on top of the previous structural layer to form another pattern. Since the thickness of the structural layer is thinner, the distance between the two patterns is significantly shortened compared to other manufacturing methods.

[0092] For example, when Faraday waves are usually used for patterning, the stable height of the liquid (that is, the height required to form a stable standing wave field) is generally greater than 0.8 mm. In this application, when patterning the assembly unit, the height of the pre-assembly liquid can be greater than or equal to the stable height, and the thickness of the structural layer can be less than the stable height. This shortens the distance between the two patterns while ensuring the patterning quality.

[0093] In the field of tissue engineering, reducing the interlayer spacing is beneficial to the connection and communication between cells in two adjacent layers (that is, the assembly units in this application), thereby facilitating the formation of tissues. Therefore, the assembly method of this application has better flexibility in constructing tissues and organs and has broad application prospects.

[0094] Exemplarily, at least one structural layer has a thickness of less than 0.5 mm.

[0095] In order to more accurately control the amount of material in the assembly unit in the next structural layer, the assembly unit can be optionally supplemented, including removing the uncured pre-assembly liquid in the assembly chamber and adding new pre-assembly liquid to the assembly chamber. By re-adding the pre-assembly liquid, the accuracy of the assembly unit is improved on the one hand, and the mixing of the assembly unit and the light-curing liquid is also facilitated on the other hand, which helps to improve the manufacturing accuracy of the multi-layer structure.

[0096] It is understandable that the parameters of different structural layers may be the same or different, for example, the amount of material of the assembly units in each structural layer and the formed pattern may be the same or different.

[0097] It should be pointed out that the curing of the bio-ink requires reaction time, and in step S300, only the bio-ink at the bottom layer is required to be stabilized, and it is not required to be completely cured. Therefore, there will be bio-ink in a semi-cured state on the upper surface of the structural layer, and the degree of curing at different positions on the upper surface may be uneven, which may cause unexpected undulations and concave-convex defects on the upper surface. These defects will affect the sedimentation effect of the assembly unit of the next layer.

[0098] To this end, optionally, the structural layer can be completely cured after removing the pre-assembly liquid and before adding new pre-assembly liquid (for example, irradiating the photosensitive bio-ink from the top downward, or heating the thermosensitive bio-ink again). Taking the photosensitive bio-ink as an example, optionally, a small amount of pre-assembly liquid can be added to the top of the structural layer once or multiple times during this process (for example, dripping), and after dripping, irradiating from the top downward for curing, thereby filling up the undulations and concave-convex defects to obtain a structural layer with a smoother upper surface. For thermosensitive bio-ink, a similar method can also be used to drip and cure multiple times on the top to obtain a structural layer with a smoother upper surface.

[0099] Alternatively, optionally, a certain amount of liquid pre-assembly liquid may be retained when the pre-assembly liquid is removed. Under the effect of fluidity, the liquid pre-assembly liquid will fill up the bumps and concave-convex defects, so that a structural layer with a smoother upper surface can be obtained when the structural layer is fully solidified later.

[0100] According to the assembly method provided in the present application, patterning is performed after the assembly units have settled to the bottom layer of the pre-assembly liquid, and then a gradual solidification method from the bottom up is adopted to selectively stabilize only a portion of the bio-ink located at the bottom layer, thereby forming a structural layer with patterned assembly units at the bottom layer of the assembly chamber. In this way, the thickness of the structural layer can be made smaller than the height of the pre-assembly liquid during patterning, thereby reducing the spacing between the assembly units located in adjacent structural layers, and constructing a multilayer structure with a low spacing.

[0101] In the multilayered tissue prepared by this method, the thickness of the single-layer structure layer (or the interlayer spacing of the assembly unit) can break through the minimum thickness limit required to form a stable standing wave field, for example, less than 0.8mm, and can reach a minimum of 0.1-0.3mm, depending on the size of the assembly unit, so it exhibits excellent biological properties, such as being more conducive to interlayer cell connection and communication. In addition, reducing the interlayer spacing can reduce the use of expensive pre-assembly fluid, and the biocompatible curable precursor used in the pre-assembly fluid is expensive, so this method can reduce the manufacturing cost of artificial tissue.

[0102] The standing wave assembly device and assembly vessel provided in the present application are suitable for implementing the assembly method provided in the present application.

[0103] For example, refer to Figure 1 and Figure 2 The standing wave assembly device embodiment of the present application includes an assembly table 110 and a curing component. The assembly table 110 is used to place an assembly carrier 120. The assembly table 110 is formed with a vertical through light hole, which is used to expose the bottom of the assembly carrier 120. The curing component includes a light source 210 and a heat source 220. The curing component is located below the light hole, and the curing component is used to cure the pre-assembly liquid in the assembly carrier 120 from the bottom.

[0104] According to the standing wave assembly device of the present application, a curing component is arranged below the assembly table 110 so that the curing component can perform photocuring or thermal curing on the pre-assembly liquid in the assembly carrier 120 from the bottom. By controlling the curing process, the pre-assembly liquid in the bottom layer can be selectively stabilized (completely or partially cured) to form a structural layer with a thickness less than that of the pre-assembly liquid, which is conducive to producing a structural layer with a thickness less than the minimum thickness required to form a stable standing wave field. Therefore, the standing wave assembly device is conducive to constructing a multi-layer structure with a low spacing.

[0105] It is understandable that the standing wave assembly device further comprises an excitation component, which is used to generate standing waves required for patterning of the assembly units.

[0106] For example, refer to Figure 1 The excitation component may include a supporting arm 310 and a transducer 320 , the assembly platform 110 is disposed on the supporting arm 310 , the supporting arm 310 is disposed on the transducer 320 , and the transducer 320 is used to generate vibration to generate standing waves in the pre-assembly liquid through the supporting arm 310 .

[0107] As mentioned above, in the present application, the standing wave can be a linear or nonlinear standing wave. The present application preferably uses nonlinear Faraday waves for biological assembly. The assembly units can gradually gather at the nodes of the Faraday standing wave field to form a pattern under the action of the hydraulic drag force mediated by the Faraday wave.

[0108] The transducer 320 may be a conventional acoustic transducer, including an exciter, a voice coil motor, a high-speed translation stage, etc. Of course, it is not excluded that other mechanical devices capable of receiving electrical signals and converting the electrical signals into stable, periodic mechanical vibration signals may be used as the transducer 320, and this application does not impose any limitation on this.

[0109] In the present application, the light source 210 and the heat source 220 may be configured in different ways, as long as they are located at the bottom and are suitable for achieving gradual curing from bottom to top.

[0110] For example, in order to reduce the loss of heat during the transmission process, refer to Figure 2 The heat source 220 may include a temperature-variable film (a heating film based on a resistance wire or a cooling film based on a semiconductor device, etc.), which is disposed at the bottom of the assembly platform 110 and covers the light-transmitting hole, so that the heat source 220 is as close as possible to the assembly carrier 120. Accordingly, the light source 210 is located below the temperature-variable film.

[0111] However, the temperature variable film will also cover the light-transmitting holes, affecting the light curing of the pre-assembly liquid. To ensure that the light is not affected, the material of the temperature variable film is preferably a transparent material so that the light can pass through the temperature variable film to irradiate the assembly carrier 120 .

[0112] Optionally, refer to Figure 1 The light source 210 uses a light box that can emit parallel light. The light box is placed under the assembly table 110. During assembly, the curing process can be regulated by adjusting the intensity of the light source 210 or by adjusting the distance between the light source 210 and the assembly table 110.

[0113] For example, in Figure 1 In the illustrated embodiment, the standing wave assembly device also includes a manipulator 510, which can clamp the light source 210, thereby changing the position of the light source 210 as needed, such as moving the light source 210 in the vertical direction, adjusting the distance between the light source 210 and the assembly table 110, or moving the light source 210 originally placed under the assembly table 110 to the top of the assembly table 110, irradiating the pre-assembly liquid from above, so that the pre-assembly liquid is completely cured.

[0114] Optionally, the standing wave assembly device also includes a sample rack 410, which is used to place test tubes 420 and pipettes 430. There can be multiple test tubes 420, which are used to hold the configured pre-assembly liquid and the waste liquid removed after solidification. The pipette 430 can accurately and quantitatively add the pre-assembly liquid to meet the needs of making a thinner structural layer.

[0115] Optionally, the robot 510 can also be used to perform operations such as pipetting, which helps to automate the multilayer structure manufacturing process.

[0116] Optionally, the standing wave assembly device further includes a buckle 130, which is movably disposed on the assembly platform 110, and is used to press and fix the assembly carrier 120, thereby preventing the assembly carrier 120 from shaking or position shifting, thereby improving the quality of patterning.

[0117] For example, refer to Figure 1 and Figure 2 The buckle 130 can be rotatably arranged on the assembly table 110, and a return spring (such as a torsion spring) is installed between the buckle 130 and the assembly table 110. The two buckles 130 are relatively arranged on both sides of the light-transmitting hole. When the assembly carrier 120 is placed, the end of the buckle 130 away from the light-transmitting hole is pressed to lift the buckle 130 and move it away from the top of the light-transmitting hole; after the assembly carrier 120 is placed, the buckle 130 is released, and under the action of the return spring, the buckle 130 rotates and presses the assembly carrier 120 from the top.

[0118] It can be understood that there are many options for the assembly carrier 120 in the present application, as long as it is suitable for containing the pre-assembly liquid and performing patterning and bottom curing.

[0119] For example, refer to Figure 3 In the illustrated embodiment, the present application discloses an assembly vessel that can be used as an assembly carrier 120. Specifically, the assembly vessel of this embodiment includes a vessel body 121 and a bottom cover 122. The vessel body 121 is formed with a vertically through assembly chamber 123. The bottom cover 122 is disposed on the vessel body 121, and the bottom cover 122 closes the bottom of the assembly chamber.

[0120] In the case of light curing, in order to meet the light transmission requirement, the material of the bottom cover 122 can be a transparent material.

[0121] In the case of thermal curing, in order to transfer heat mainly to the pre-assembly liquid through the bottom cover 122 , the thermal conductivity of the bottom cover 122 should be greater than the thermal conductivity of the vessel body 121 .

[0122] Preferably, the vessel body 121 is made of insulating material or other material that is difficult to conduct heat, and the bottom cover 122 is made of material with good thermal conductivity. In this way, when heated by the heat source 220, it is difficult for heat to be transferred from the bottom cover 122 to the vessel body 121. Therefore, most of the heat is transferred from the bottom cover 122 to the pre-assembly liquid, ensuring that the pre-assembly liquid is gradually solidified from bottom to top, avoiding solidification from outside to inside under the heating of the vessel body 121, which affects the production of the multi-layer structure.

[0123] Optionally, the assembly vessel is placed in the light-transmitting hole, and the outer diameter of the vessel body 121 is smaller than the inner diameter of the light-transmitting hole, so that a gap is formed between the vessel body 121 and the light-transmitting hole. The air in the gap can produce a heat-insulating effect, thereby preventing the heat source 220 from indirectly heating the vessel body 121 by heating the assembly table 110.

[0124] Of course, the assembly vessel is only one of the optional ways to assemble the carrier 120. In other possible embodiments, a culture plate (e.g., a 6-, 12-, 48-, or 96-well plate) can also be used as the assembly carrier 120. The advantage of the culture plate is that it can meet the needs of assembling multiple multilayer structures at the same time, thereby achieving high-throughput Faraday wave biological assembly.

[0125] Other feasible assembly carriers 120 can refer to the relevant prior art and will not be described in detail here.

[0126] The present application further illustrates the assembly method provided by the present application through the following specific embodiments. It is worth noting that the following description is only an exemplary description and not a specific limitation of the present application. This specific embodiment can also be replaced by the above-mentioned corresponding technical features or combined with the above-mentioned technical features.

[0127] If the specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. If the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

[0128] Embodiment 1:

[0129] (1) Construction of assembly platform

[0130] Example 1 uses Figure 1 , Figure 2 , Figure 3 The standing wave assembly device and assembly vessel shown in the figure are cured by light curing. The light source 210 uses 405 nm power of 25 mW / cm 2 Blue light source.

[0131] (2) Preparation of pre-assembly solution

[0132] GelMA was placed in a 0.25% (w / v) LAP initiator standard solution and stirred thoroughly to dissolve, obtaining a 8% (w / v) GelMA solution. Chitosan powder (as an assembly unit, used to simulate cells in the experiment) was dispersed in the GelMA solution at a concentration of 25 mg / mL to obtain a pre-assembly solution.

[0133] (3) Assembly of multi-layer structures

[0134] The uniformly mixed pre-assembly liquid is added into the assembly chamber 123 .

[0135] After the chitosan settles to the bottom layer of the pre-assembly liquid, it is driven by a Faraday wave with a frequency of 20 Hz to move to form a cross patterned structure.

[0136] Turn on the light source 210 and irradiate from bottom to top for 30 seconds to crosslink the pre-assembly liquid near the bottom of the assembly chamber (that is, the pre-assembly liquid at the bottom layer) and transform it into a stable state, fix the obtained patterned structure, and obtain the first structural layer ( Figure 5 (a)

[0137] 1 / 2 of the pre-assembly liquid volume of the assembly chamber is sucked from the top of the assembly chamber, and then the light source 210 is clamped by the robot 510 and moved to the top of the assembly table 110, and irradiated from top to bottom for 2 minutes to completely cross-link the remaining pre-assembly liquid in the assembly chamber.

[0138] The pre-assembly liquid containing the dispersed assembly units was added to the assembly chamber to a volume of 1 / 2 of the assembly chamber. After the assembly units settled to the bottom of the pre-assembly liquid, the chitosan powder was driven to move by a Faraday wave with a frequency of 20 Hz to form a cross patterned structure.

[0139] Then, the pre-assembled liquid in the chamber is completely cross-linked by irradiating with blue light from top to bottom for 2 minutes to obtain the second structural layer, thus completing the production of a low-spacing multi-layer (here double-layer) structure. Figure 5 Parts b and c, where b is a top view and c is a side view).

[0140] Embodiment 2:

[0141] Example 2 is based on Example 1, except that the Faraday wave driving frequency is 25 Hz. The remaining steps are the same as Example 1, and the results are as follows: Figure 6 shown. Figure 6 Part a in the middle shows a top view of the first structural layer. Figure 6 Parts b and c show the top view and side view of the multilayer structure.

[0142] Embodiment 3:

[0143] Example 3 is based on Example 1, except that the Faraday wave driving frequency is 50 Hz. The remaining steps are the same as Example 1, and the results are as follows: Figure 7 shown. Figure 7 Part a in the middle shows a top view of the first structural layer. Figure 6 Parts b and c show the top view and side view of the multilayer structure.

[0144] Embodiment 4:

[0145] Example 4 is based on Example 1, except that the Faraday wave driving frequency is 56 Hz. The remaining steps are the same as those of Example 1. The results are as follows: Figure 8 shown. Figure 8 Part a in the middle shows a top view of the first structural layer. Figure 8 Parts b and c show the top view and side view of the multilayer structure.

[0146] Embodiment 5:

[0147] Embodiment 5 is based on embodiment 1, and the difference is as follows:

[0148] First, the pre-assembly liquid after mixing was added into the assembly chamber with a thickness of 2 mm. After the chitosan settled to the bottom of the pre-assembly liquid, it was driven to move by a Faraday wave with a frequency of 48 Hz to form a double-layer concentric circle patterned structure. The light source 210 was turned on and irradiated from bottom to top for 30 seconds to cross-link the pre-assembly liquid near the bottom of the assembly chamber, fix the obtained patterned structure, and obtain the first structural layer ( Fig. 9 3 / 4 of the volume of the pre-assembly liquid in the assembly chamber is drawn from the top of the assembly chamber, and then a light source 210 is used to illuminate the top of the assembly chamber from top to bottom for 2 minutes to completely cross-link the remaining pre-assembly liquid in the chamber.

[0149] Next, add 3 / 4 of the volume of the assembly chamber to the pre-assembly liquid with the assembly units dispersed therein. After the assembly units settle to the bottom of the pre-assembly liquid, the chitosan powder is driven to move by a Faraday wave with a frequency of 50 Hz to form a double-layer concentric circle patterned structure. Then, the double-layer concentric circle patterned structure is stabilized by irradiating with blue light from bottom to top for 2 minutes to obtain the second structural layer. 1 / 2 of the volume of the pre-assembly liquid is drawn from the top of the assembly chamber, and then a 405nm blue light source is used at the top of the assembly chamber to irradiate from top to bottom for 2 minutes to completely cross-link the remaining pre-assembly liquid in the chamber.

[0150] Finally, the pre-assembly liquid with dispersed assembly units was added to 1 / 2 of the assembly chamber volume. After the assembly units settled to the bottom of the pre-assembly liquid, the chitosan powder was driven to move by a Faraday wave with a frequency of 50 Hz to form a double-layer concentric circle patterned structure. Then, blue light was irradiated from top to bottom for 2 minutes to completely cross-link the pre-assembly liquid in the chamber, completing the production of a multi-layer (here three-layer) structure with a low spacing (0.5 mm / layer).

[0151] The results are as follows Fig. 9 shown. Fig. 9 Part a in the middle shows a top view of the first structural layer. Fig. 9 Part b shows a top view of the second structural layer. Fig. 9 Parts c and d show the top view and side view of the multilayer structure.

[0152] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "illustrative embodiments", "examples", "specific examples", or "some examples" means that the specific features, structures, materials, or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present application. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described may be combined in any one or more embodiments or examples in a suitable manner.

[0153] Although the embodiments of the present application have been shown and described, those skilled in the art will appreciate that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present application, and that the scope of the present application is defined by the claims and their equivalents.

Claims

1. A standing wave assembly device, characterized in that: include: An assembly platform, the assembly platform is used to place an assembly carrier, the assembly platform is formed with a vertically penetrating light-transmitting hole, and the light-transmitting hole is used to expose the bottom of the assembly carrier; A curing component, comprising a light source and a heat source, is located below the light-transmitting hole, and is used to cure the pre-assembly liquid in the assembly carrier from the bottom.

2. The standing wave assembly device according to claim 1, characterized in that: The heat source comprises a temperature-variable film, which is arranged at the bottom of the assembly platform and covers the light-transmitting hole. The temperature-variable film is made of transparent material, and the light source is located below the temperature-variable film.

3. The standing wave assembly device according to claim 2, characterized in that: The standing wave assembly device includes an excitation component, which includes a supporting arm and a transducer. The assembly platform is arranged on the supporting arm, and the supporting arm is arranged on the transducer. The transducer is used to generate vibration to generate standing waves in the pre-assembly liquid through the supporting arm.

4. The standing wave assembly device according to claim 1, characterized in that: The standing wave assembly device comprises a buckle, which is movably arranged on the assembly platform and is used to press and fix the assembly carrier.

5. An assembly vessel, characterized in that: The invention comprises a vessel body and a bottom cover, wherein the vessel body is formed with a vertically through assembly chamber, the bottom cover is arranged on the vessel body, the bottom cover closes the bottom of the assembly chamber, the material of the bottom cover is a transparent material and / or the thermal conductivity of the bottom cover is greater than the thermal conductivity of the vessel body.

6. An assembly method, characterized in that: include: Adding a pre-assembly liquid into the assembly chamber, wherein the pre-assembly liquid includes an assembly unit and a biological ink, wherein the biological ink has photosensitivity and / or temperature sensitivity; After the assembly units are deposited to the bottom layer of the pre-assembly liquid, a standing wave is applied to make the assembly units aggregate in a pattern; Stabilizing the bottom layer of the bio-ink from the bottom to fix the patterned assembly units to obtain a first structural layer; The pre-assembly liquid is replenished, and according to the above process, the next structural layer is manufactured based on the first structural layer.

7. The assembly method according to claim 6, characterized in that: Replenishing the pre-assembly liquid includes removing the pre-assembly liquid located on the upper layer of the assembly chamber and adding new pre-assembly liquid into the assembly chamber.

8. The assembly method according to claim 7, characterized in that: After removing the pre-assembly liquid and before adding new pre-assembly liquid, the structural layer is completely cured.

9. The assembly method according to claim 6, characterized in that: When patterning the assembly unit, the height of the pre-assembly liquid is greater than or equal to the minimum thickness required to form a stable standing wave field, the thickness of the structural layer is less than the minimum thickness required to form a stable standing wave field, and a multi-layer structure is constructed.

10. Application of the assembly method according to any one of claims 6 to 9 in any one of the following: A1), constructing an engineered bone-cartilage interface structure; A2), constructing the skin layered structure; A3) Building vascular structure; A4), construct cartilage, skin or vascular disease models; A5) preparing tissue repair materials.

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