3D printing platform integrated with holographic optical tweezers, and bionic tissue construction method

Abstract

The present invention relates to the technical field of tissue engineering, and in particular to a 3D printing platform integrated with holographic optical tweezers, and a bionic tissue construction method. The printing platform comprises: a holographic optical tweezer module configured to form an optical trap array in a focal region of an objective lens of the holographic optical tweezer module; material barrels configured to store biological materials and capable of moving between a storage position and a working position; a printing base capable of moving between a material loading station and an optical tweezer manipulation station; an object stage mounted on the printing base and capable of moving in horizontal and vertical directions; a photocuring device fixed on the object stage and configured to emit light to induce the biological materials to be cured; and a cell culture plate fixed on the object stage and stacked on the photocuring device, wherein the cell culture plate has a printing chamber and a cell chamber, and the printing chamber and the cell chamber are in communication with each other by means of a channel. A macroscopic ordered three-dimensional structure can be constructed, and micro-scale organisms inside the macroscopic three-dimensional structure are also ordered and patterned.

Description

A 3D printing platform integrating holographic optical tweezers and a biomimetic tissue construction method Technical Field

[0001] This invention relates to the field of tissue engineering technology, and in particular to a 3D printing platform integrating holographic optical tweezers and a biomimetic tissue construction method. Background Technology

[0002] Constructing complex and diverse tissue structures and geometric shapes to simulate real human tissues (organs) and obtain biomimetic tissues (organs) with complete functionality can help reduce rejection reactions, promote cell adhesion, and enhance integration with surrounding tissues. This is crucial for the research and application of artificial organ modules in tissue engineering and regenerative medicine, and it also represents a current challenge.

[0003] 3D printing, through computer-aided layer-by-layer deposition, positions, stacks, and assembles living cells / cell spheres and biomaterials into specialized three-dimensional structures to create living tissues and organoids for tissue engineering, regenerative medicine, pharmacokinetics, cancer research, and other biological studies. 3D printing can construct relatively macroscopic (centimeter to millimeter) spatial structures (shape and size) of tissues (organs), supporting three-dimensional cell growth and maintaining the three-dimensional structure, offering advantages in spatial positioning and structural maintenance. Biomanufacturing also involves spatial control of objects at the micrometer and sub-micrometer scale (cells, cell spheres, microcarriers, etc.), but current 3D printing technology, due to limitations in precision and resolution, is somewhat insufficient in micrometer-scale control, making it difficult to achieve spatial positioning of minute elements within macroscopic three-dimensional structures. Therefore, it is necessary to combine other technologies to assist in precise control at the microscopic level within the printed structure, thereby achieving precise biomimetic tissue and organ construction.

[0004] Optical tweezers are tools that use the force generated by a laser beam to manipulate particles, enabling high-precision, low-damage, and non-contact capture and manipulation of biological organisms. The basic principle of optical tweezers is that a focused laser beam forms a three-dimensional optical trap, exerting a force on the object—a force known as optical radiation pressure. This pressure holds the object within the trap, allowing manipulation of objects ranging from micrometers to nanometers in size. The magnitude of this optical radiation pressure is typically in the fN to pN range, making optical tweezers a promising candidate for applications in the biomedical field. Currently, holographic optical tweezers are used for cell manipulation, transfer, and sorting of small cell samples. Optical tweezers technology offers advantages such as non-contact elastic control of biological samples, no mechanical damage, and aseptic operation. More importantly, it can directly manipulate micro- to nanometer-sized objects, such as stem cells (10-30 micrometers). However, cell assembly based on optical tweezers technology cannot maintain stability over long periods, and the manipulated size is limited to a small scale, preventing the construction of larger tissues (organs). Furthermore, capture and manipulation are limited to the planar direction (X, Y axes), making control difficult in the Z-axis direction.

[0005] Therefore, effectively combining 3D printing and optical tweezers technology, leveraging their respective strengths and compensating for their weaknesses, is of great significance for tissue engineering and organ manufacturing. Summary of the Invention

[0006] In view of the above-mentioned shortcomings and deficiencies of the prior art, the present invention provides a 3D printing platform integrating holographic optical tweezers and a biomimetic tissue construction method, which can construct macroscopic three-dimensional structures, and achieve ordering and patterning of biological organisms inside the macroscopic three-dimensional structures.

[0007] To achieve the above objectives, the main technical solutions adopted by the present invention include:

[0008] In a first aspect, the present invention provides a 3D printing platform integrating holographic optical tweezers, comprising:

[0009] A holographic optical tweezers module is used to modulate a laser beam and form an optical trap array in the focal region of the objective lens of the holographic optical tweezers module;

[0010] The material container is used to store biological materials and can be moved between the storage location and the working location;

[0011] The printing base can move between the feeding station and the optical tweezers control station; at the feeding station, the printing base is located below the material cylinder, and at the optical tweezers control station, the printing base is located below the objective lens.

[0012] The stage, mounted on the printing base, can move horizontally to assist in the movement of the optical trap;

[0013] A photopolymerization device, fixed on a stage, is used to emit light to induce the curing of biological materials;

[0014] Cell culture plates are fixed on a stage and stacked on a photocuring device; the cell culture plates have a printing chamber and a cell chamber, which are connected by a channel;

[0015] The printing base is located at the feeding station. The barrel moves to the working position, and the extrusion nozzle of the barrel extends into the cell chamber of the cell culture plate. The printing base is located at the optical tweezers manipulation station. The optical trap array captures organisms in the cell chamber. The stage moves horizontally, allowing the organisms captured by the optical trap array to move through the channel to the printing chamber.

[0016] Optionally, the holographic optical tweezers module includes a laser source, which is a near-infrared laser source, and the objective lens is selected to be 40x, 50x or 100x near-infrared light-transmitting objective lens.

[0017] Optionally, the 3D printing platform integrating holographic optical tweezers also includes a first power mechanism and a second power mechanism; the first power mechanism is connected to the printing base and drives the printing base to move between the feeding station and the optical tweezers control station; the second power mechanism is mounted on the printing base and connected to the stage and drives the stage to move in the horizontal direction.

[0018] Optionally, the first power mechanism drives the printing base to move along a straight line between the feeding station and the optical tweezers control station, or the first power mechanism drives the printing base to rotate from the feeding station to the optical tweezers control station.

[0019] Optionally, the 3D printing platform integrating holographic optical tweezers also includes a barrel moving mechanism and a pneumatic pump; the barrel moving mechanism is connected to the barrel drive, and the barrel moving mechanism drives the barrel to move sequentially between the storage position, the intermediate position and the working position. The barrel moving mechanism drives the barrel to move horizontally from the storage position to the intermediate position, and the barrel moving mechanism drives the barrel to move vertically from the intermediate position to the working position; the pneumatic pump is connected to the barrel, and the air pressure generated by the pneumatic pump controls the extrusion of biomaterial from the discharge needle of the barrel; the stage can also move vertically for focusing of the objective lens.

[0020] Optionally, the light source of the photocuring device is an array of semiconductor light-emitting units laid out in the horizontal direction. The size of the semiconductor light-emitting units is 1~100μm, and each semiconductor light-emitting unit can be driven to emit light independently.

[0021] Optionally, in the cell culture plate, in the vertical direction, the channel is higher than the bottom surface of the cell chamber and the bottom surface of the printing chamber, and the bottom surface of the cell chamber is connected to the channel by a slope.

[0022] Secondly, the present invention provides a biomimetic tissue construction method, which uses the 3D printing platform with integrated holographic optical tweezers as described above to construct biomimetic tissue, and includes the following steps:

[0023] Preparation steps: Load the biological materials required for printing into the material cylinder and fix the cell culture plate on the stage;

[0024] Material feeding process: The printing base moves to the material feeding station, the material cylinder moves to the working position, and the extrusion nozzle of the material cylinder extends into the cell chamber of the cell culture plate to extrude biological material into the cell chamber; after the material feeding is completed, the material cylinder first moves to the storage position, and then the printing base moves to the optical tweezers control station;

[0025] Single-layer bioprinting process: The printing base is located at the optical tweezers control station. The optical trap array generated by the holographic optical tweezers module captures the organism in the cell chamber. The stage moves horizontally, allowing the organism captured by the optical trap array to move through the channel to the printing chamber for arrangement, until a single-layer ordered array of organisms is formed in the printing chamber. The photocuring device performs photo-pre-crosslinking on the organism array to form a single-layer micron-scale ordered organism.

[0026] The single-layer biological printing process is repeated, and single-layer micron-scale ordered biological bodies are stacked vertically to obtain a tissue model. The tissue model is then cross-linked by light irradiation using a photocuring device to obtain a biomimetic tissue.

[0027] Optionally, the preparation process also includes: leveling the printing base and stage, and positioning the cell culture plate.

[0028] Optionally, in the single-layer bio-printing process, after the bio-array is formed in the printing chamber, the printing base moves to the feeding station, the barrel moves to the working position, and the extrusion nozzle of the barrel extends into the printing chamber to suspend and print supporting biomaterials at specific edge positions of the bio-array, forming a framework. Then, a photocuring device performs photo-pre-crosslinking on the bio-array to form a single-layer bio-array; or...

[0029] In the single-layer bio-printing process, after the bio-array is pre-crosslinked by light irradiation in the photocuring device, the printing base moves to the feeding station, the barrel moves to the working position, and the extrusion nozzle of the barrel extends into the printing chamber to suspend and print supporting biological materials at specific edge positions of the bio-array to form a framework and obtain a single-layer bio-array.

[0030] The beneficial effects of this invention are:

[0031] The present invention provides an integrated holographic optical tweezers 3D printing platform and a biomimetic tissue construction method. The printing base moves to the feeding station, the material cylinder moves to the working position, and the extrusion nozzle of the material cylinder extends into the cell chamber of the cell culture plate to extrude biological material into the cell chamber. The printing base moves to the optical tweezers control station, and the optical trap array generated by the holographic optical tweezers module captures organisms in the cell chamber. The stage moves horizontally, so that the organisms captured by the optical trap array move through the channel to the printing chamber for arrangement until a single-layer ordered array of organisms is formed in the printing chamber. The photocuring device performs photo-pre-crosslinking on the organism array to form a single-layer organism. The above process is repeated to stack the single-layer organisms in the vertical direction, which can obtain biomimetic tissue with macroscopic three-dimensional structure. As can be seen, the 3D printing platform and biomimetic tissue construction method integrating holographic optical tweezers provided by this invention combine the advantages of 3D printing in constructing stable three-dimensional structures at the macroscopic (cm / mm) scale and holographic optical tweezers in assembling cells at the microscopic (micrometer / nanometer) scale. It can construct macroscopically stable three-dimensional structures, and the organisms inside the macroscopic three-dimensional structures are ordered and patterned, further simulating human tissue, so that the printed tissues (organs) have more complete organ functions. Attached Figure Description

[0032] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used together with the following detailed description to explain the invention, but do not constitute a limitation thereof. In the drawings:

[0033] Figure 1 is a schematic diagram of the holographic optical tweezers module in a specific embodiment;

[0034] Figure 2 is a schematic diagram of the optical trap array in a specific embodiment;

[0035] Figure 3 is a schematic diagram of the 3D printing platform integrating holographic optical tweezers in a specific embodiment;

[0036] Figure 4 is a schematic diagram of the structure in a specific embodiment where the printing base moves along a straight line between the feeding station and the optical tweezers control station.

[0037] Figure 5 is a schematic diagram of the structure of the printing base rotating and moving between the feeding station and the optical tweezers control station in a specific embodiment.

[0038] Figure 6 is a top view of the stage equipped with the photocuring device in a specific embodiment;

[0039] Figure 7 is a cross-sectional schematic diagram of the stage equipped with the photocuring device in a specific embodiment;

[0040] Figure 8 is a schematic diagram of the cell culture plate in a specific embodiment;

[0041] Figure 9 is a top view of the organ-on-a-chip in Example 1;

[0042] Figure 10 is a schematic diagram of a single layer of cardiomyocytes, a single layer of endothelial cells, and a single layer of fibroblasts in Example 1.

[0043] Figure 11 is a schematic diagram of the tissue model in Example 1;

[0044] Figure 12 is a schematic diagram of the structure of the first monolayer cell, the second monolayer cell, and the third monolayer cell in Example 2.

[0045] Explanation of reference numerals in the attached figures

[0046] 11: Laser source; 12: Diffraction element; 13: Lens; 14: Dichroic mirror; 15: Filter; 16: Mirror; 17: Objective lens; 18: Beamconer module; 19: Illumination source; 20: Camera;

[0047] 31: Printing base; 32: Material cylinder; 33: Stage; 34: Photopolymerization device;

[0048] 41: Temperature control board; 42: Light-emitting board;

[0049] 5: Cell culture plates;

[0050] 51: Cell chamber; 52: Printing chamber; 53: Slope; 54: Channel. Detailed Implementation

[0051] To better explain and facilitate understanding of the present invention, a detailed description of the invention is provided below with reference to the accompanying drawings and specific embodiments. In this document, the "vertical direction" refers to the direction of gravity.

[0052] To clearly introduce the integrated holographic optical tweezers 3D printing platform provided by this invention, the holographic optical tweezers system and the multi-barrel 32 extrusion 3D printing system will be described below.

[0053] Holographic optical tweezers technology enables precise capture and manipulation of multiple tiny objects in three-dimensional space. Its working principle is to use holographic optical elements (diffraction element 12) to modulate a laser beam with computer assistance, so that the laser beam forms a desired light field distribution, i.e., an optical trap array, in the focal area of ​​objective lens 17.

[0054] As shown in Figure 1, the holographic optical tweezers system of this invention includes a laser source 11, a spatial light modulator (SLM) (containing a diffraction element 12), other optical components (lens 13, dichroic mirror 14, filter 15, reflector 16, objective lens 17, beam shrinking module 18), detectors (illumination source 19, camera 20, etc.), and a printing base 31. The laser source 11 is the energy source for the holographic optical tweezers, providing a high-intensity, monochromatic laser beam for forming optical trap arrays and manipulating particles. The SLM is the core component of the holographic optical tweezers, capable of converting a single laser beam into a computer-programmed optical trap pattern, enabling the simultaneous capture and manipulation of multiple particles. Computer programming can design linear, square, semi-circular, circular, and other horizontal optical trap arrays (as shown in Figure 2), as well as the movement trajectory of the optical trap array, as needed. The SLM is an existing and mature optical device; its internal structure and principles will not be described further. Other optical components, such as lens 13, dichroic mirror, filter 15, reflector 16, and beam shortening module 18, are essential parts of the entire optical path system, used for focusing, filtering, and changing the beam direction. Detectors, such as illumination source 19 and camera 20, are used to monitor and provide feedback on the position and movement of the organism. In Figure 1, the red arrow indicates the optical path of the laser beam, used to form the optical trap array, and the blue arrow indicates the optical path of the LED illumination light, used to monitor the position and movement of the organism.

[0055] The multi-tube 32-extrusion 3D printing system enables the orderly stacking of various materials in three-dimensional space to form supporting and fixed structures. Its working principle involves using controlled pressure in conjunction with a precisely positioned robotic arm to stack bio-ink containing cells, cell spheres, microcarriers, and biological materials into a three-dimensional structure according to a design model.

[0056] As shown in Figure 3, the integrated holographic optical tweezers 3D printing platform provided by the present invention includes: a holographic optical tweezers module for modulating a laser beam to form an optical trap array in the focal area of ​​the objective lens 17 of the holographic optical tweezers module; a material cylinder 32 for storing biological materials and capable of moving between a storage position and a working position; a printing base 31 capable of moving between a feeding station and an optical tweezers control station; at the feeding station, the printing base 31 is located below the material cylinder 32, and at the optical tweezers control station, the printing base 31 is located below the objective lens 17; a stage 33 mounted on the printing base 31 and capable of moving horizontally; a photopolymerization device 34 fixed on the stage 33 for emitting light to induce the curing of biological materials; and a cell culture plate 5 fixed on the stage 33 and stacked on the photopolymerization device 34; the cell culture plate 5 has a printing chamber 52 and a cell chamber 51, which are connected by a channel 54.

[0057] The printing base 31 is located at the feeding station, the material cylinder 32 moves to the working position, and the extrusion nozzle of the material cylinder 32 extends into the cell chamber 51 of the cell culture plate 5; the printing base 31 is located at the optical tweezers manipulation station, the optical trap array captures organisms in the cell chamber 51, and the stage 33 moves horizontally, so that the organisms captured by the optical trap array are moved to the printing chamber 52 through the channel 54.

[0058] In this integrated holographic optical tweezers 3D printing platform, the printing base 31 moves to the feeding station, the material cylinder 32 moves to the working position, and the extrusion nozzle of the material cylinder 32 extends into the cell chamber 51 of the cell culture plate 5 to extrude biological material into the cell chamber 51. The printing base 31 moves to the optical tweezers control station, and the optical trap array generated by the holographic optical tweezers module captures organisms in the cell chamber 51. The stage 33 moves horizontally, so that the organisms captured by the optical trap array are moved through the channel 54 to the printing chamber 52 for arrangement until a single-layer orderly array of organisms is formed in the printing chamber 52. The photocuring device 34 performs photo-pre-crosslinking on the organism array to form a single-layer organism. By repeating the above process and stacking the single-layer organisms in the vertical direction, biomimetic tissues with macroscopic three-dimensional structures can be obtained. As can be seen, the 3D printing platform with integrated holographic optical tweezers provided by this invention combines the advantages of 3D printing in constructing stable three-dimensional structures at the macroscopic (cm / mm) scale and holographic optical tweezers in assembling cells at the microscopic (micrometer / nanometer) scale. It can construct macroscopically stable three-dimensional structures, and the organisms inside the macroscopic three-dimensional structures are ordered and patterned, further simulating human tissue, so that the printed tissues (organs) have more complete organ functions.

[0059] It should be noted that the structure of the holographic optical tweezers module is the same as that of the holographic optical tweezers system described above, and will not be repeated here.

[0060] Typically, holographic optical tweezers systems are equipped with a 540nm laser source 11. However, high-power visible light lasers can damage biological tissue. Preferably, in this invention, the laser source 11 of the holographic optical tweezers module is a near-infrared laser source 11, emitting near-infrared lasers with wavelengths above 1064nm. Near-infrared lasers in the 1064nm and longer wavelength bands have higher power (greater than 1W), resulting in more stable laser capture and manipulation. Furthermore, lasers in this band do not produce strong thermal effects, causing less damage to tissues. Accordingly, all optical components of the holographic optical tweezers module are selected to be compatible with the near-infrared band.

[0061] Preferably, the objective lens 17 of the holographic optical tweezers module is selected to be 40x, 50x, or 100x near-infrared light transmittance. In this way, the working distance of the objective lens 17 is relatively long, which can reserve a large (vertical) space between the objective lens 17 and the printing base 31, which is beneficial for accommodating thicker cell culture plates 5, thereby enabling the printing of large-sized biomimetic tissues, and ensuring that the printing base 31 will not collide with the objective lens 17 during the movement.

[0062] Preferably, the 3D printing platform integrating holographic optical tweezers also includes a barrel moving mechanism and a pneumatic pump. The barrel moving mechanism is drively connected to the barrel 32, driving the barrel 32 to move sequentially between a receiving position, a middle position, and a working position. Specifically, the barrel moving mechanism drives the barrel 32 horizontally from the receiving position to the middle position, and vertically from the middle position to the working position. Thus, the barrel moving mechanism can control the barrel 32 to first move horizontally to the middle position to extend from the receiving position, then vertically to the working position to position itself within the cell cavity or printing cavity of the cell culture plate. After the barrel 32 has extruded the biological material, it first moves vertically to the middle position, then horizontally to the receiving position to retract back to its original position. The pneumatic pump is connected to the barrel 32, and the air pressure generated by the pneumatic pump can extrude the biological material from the discharge needle of the barrel 32. In this way, the biomaterial in the barrel 32 is extruded, and the pneumatic pump can be used to control the speed and amount of biomaterial extruded from the barrel 32.

[0063] The biomaterials stored in the barrel 32 can be fluid bio-inks, hydrogels, cells, 3D cell spheres, and micro / nano particles, etc.

[0064] Furthermore, the barrel 32 has a temperature control function to maintain the performance of the biomaterial stored inside the barrel 32.

[0065] Generally, a 3D printing platform integrating holographic optical tweezers includes at least two barrels 32, each of which can store different types of biomaterials. This allows for the orderly arrangement and stacking of various biomaterials during the printing process, enabling the construction of more complex biomimetic tissues.

[0066] As an example, the barrel moving mechanism is a robotic arm.

[0067] Preferably, the 3D printing platform integrating holographic optical tweezers further includes a first power mechanism and a second power mechanism; the first power mechanism is connected to the printing base 31 and drives the printing base 31 to move between the feeding station and the optical tweezers control station; the second power mechanism is mounted on the printing base 31 and connected to the stage 33 and drives the stage 33 to move in the horizontal and vertical directions. The movement of the printing base 31 between the feeding station and the optical tweezers control station is a large-scale movement on the centimeter level, generally within a range of 10-20 cm, with a minimum movement distance of 1 cm per operation. To enable the biological organism captured by the optical trap array to move through the channel 54 to the stage 33 of the printing chamber 52, nanoscale small-range movement can be achieved. The minimum movement distance per operation is 50nm, and the maximum movement distance after multiple operations is 15mm. The movement range is small but the precision is high. Therefore, by using two power mechanisms to drive the printing base 31 and the stage 33 respectively, a low-precision power mechanism can be selected for the printing base 31 and a high-precision power mechanism can be selected for the stage 33. This is easy to implement, and the structure is simple and low in cost.

[0068] As shown in Figure 4, the first power mechanism drives the printing base 31 to move horizontally along a straight line between the feeding station and the optical tweezers control station. Alternatively, as shown in Figure 5, the first power mechanism drives the printing base 31 to rotate horizontally about an axis from the feeding station to the optical tweezers control station. Further, the first power mechanism drives the printing base 31 to rotate horizontally by 90° about an axis from the feeding station to the optical tweezers control station.

[0069] Preferably, the stage 33 is also capable of moving vertically. Inside the printing chamber 52, as the biomaterial accumulates upwards, the working surface (i.e., the surface where the biomaterial accumulates) gradually deviates from the focal plane of the objective lens 17, resulting in unclear imaging of the detector of the holographic optical tweezers module, which in turn cannot guide the subsequent printing of the biomaterial based on the imaging. By setting the stage 33 to be able to move vertically, as the biomaterial accumulates upwards, the stage 33 gradually sinks, which can keep the working surface continuously and stably at the focal plane of the objective lens 17.

[0070] Furthermore, the second power mechanism drives the platform 33 to move in the vertical direction.

[0071] As shown in Figures 6 and 7, in this invention, the stage 33 has a cavity in the middle, and the photocuring device 34 is embedded in the cavity. This results in a compact structure and high space utilization.

[0072] It should be noted that the photopolymerization device can also provide white light illumination, which can be used to observe the morphology of cells and tissues under bright field conditions.

[0073] Preferably, the light source of the photopolymerization apparatus 34 is a horizontally arranged array of semiconductor light-emitting units, each with a size of 1~100μm, and each unit can be independently driven to emit light. This allows for more precise photopolymerization of the model within the printing chamber 52. Furthermore, the emission of the semiconductor light-emitting units is tunable within the ultraviolet-visible light range of 250~750nm. As an example, the semiconductor light-emitting units are micro-LEDs.

[0074] Preferably, the photocuring device 34 includes a temperature control plate 41 and a light-emitting plate 42 stacked sequentially from top to bottom. The cell culture plate 5 is stacked on the temperature control plate 41, which is made of transparent material. A temperature sensor and a heating probe are installed inside the temperature control plate 41. Thus, by setting the temperature control plate 41, while ensuring that the light from the light-emitting plate 42 induces photocuring, the temperature of the biomaterial within the cell culture plate 5 can be regulated, thereby controlling the mechanical properties of the biomaterial and facilitating printing. As an example, the temperature control plate 41 is made of quartz glass, which has high light transmittance.

[0075] As shown in Figure 8, in the cell culture plate 5, vertically, the channel 54 is higher than the bottom surface of the cell chamber 51 and the bottom surface of the printing chamber 52. The bottom surface of the cell chamber 51 is connected to the channel 54 via a slope 53. Since the optical trap captures cells, it can only generate force in the horizontal direction to restrict the cells. Therefore, the slope 53 connecting the bottom surface of the cell chamber 51 and the bottom surface of the channel 54 allows cells to climb from the cell chamber 51 onto the channel 54 and enter the printing chamber 52. The channel 54 being higher than the bottom surfaces of the cell chamber 51 and the printing chamber 52 prevents the biomaterial in the cell chamber 51 from naturally flowing into the printing chamber 52. As an example, the cell culture plate 5 is an organ-on-a-chip or a cell well plate.

[0076] Furthermore, the organ-on-a-chip has a printing chamber 52 and at least two cell chambers 51, both of which are connected to the printing chamber 52 via channels 54. The presence of at least two cell chambers 51 facilitates the printing of biomimetic tissues with complex structures.

[0077] The cell culture plate 5 is made of highly transparent quartz glass, polymethyl methacrylate (PMMA), or polydimethylsiloxane (PDMS).

[0078] Based on the aforementioned 3D printing platform integrating holographic optical tweezers, this invention also provides a biomimetic tissue construction method, comprising the following steps:

[0079] Preparation steps: Load the biological materials required for printing into the material cylinder 32, and install the cell culture plate 5 onto the stage 33.

[0080] Material feeding process: The printing base 31 moves to the material feeding station, the material cylinder 32 moves to the working position, the extrusion nozzle of the material cylinder 32 extends into the cell chamber 51 of the cell culture plate 5, and extrudes biological material into the cell chamber 51; after the material feeding is completed, the material cylinder 32 first moves to the storage position, and then the printing base 31 moves to the optical tweezers control station.

[0081] Single-layer bioprinting process: The printing base 31 is located at the optical tweezers control station. The optical trap array generated by the holographic optical tweezers module captures the organism in the cell chamber 51. The stage 33 moves horizontally, so that the organism captured by the optical trap array is moved through the channel 54 to the printing chamber 52 for arrangement, until a single-layer ordered array of organisms is formed in the printing chamber 52. The photocuring device 34 performs photo-pre-crosslinking on the organism array to form a single-layer micron-scale ordered organism.

[0082] The single-layer biological printing process is repeated, and single-layer micron-scale ordered biological bodies are stacked vertically to obtain a tissue model. The photocuring device 34 performs overall cross-linking of the tissue model by light irradiation to obtain biomimetic tissue.

[0083] The preparation process also includes leveling the printing base and stage 33, and positioning the cell culture plate.

[0084] In the process of repeating the single-layer biological body printing process, if the biological material in the cell chamber 51 is exhausted, a feeding process is performed to replenish the biological material in the cell chamber 51, and then the single-layer biological body printing process is repeated.

[0085] Generally, the bio-ink required for printing is loaded into cartridge 32. Bio-ink A is typically prepared by mixing organisms with diameters ranging from 15 to 100 micrometers, such as cells, cell spheres, organoids, and microcarriers, with a bio-media. The bio-media is a highly fluid, transparent, photosensitive material (natural, synthetic, or composite). The fluidity of the bio-media allows the light trap to move stably within the ink, and the organisms can briefly maintain their spatial position after the light trap disappears. As a photosensitive material, the bio-media possesses certain mechanical properties after photocuring, enabling it to maintain the array of organisms after the optical tweezers are removed. Suitable bio-media include natural hydrogels such as alginate, collagen, gelatin, and fibroin.

[0086] Bio-ink A needs to provide a suitable growth environment for the organisms, and its physicochemical properties determine the survival and proliferation of the organisms, so its rigidity should not be too high. However, the vertical stacking of organisms requires rigid materials for support; otherwise, excessive compression due to gravity will occur between the organisms, which is extremely detrimental to their growth and the construction of large-sized tissues. Therefore, bio-ink B, used for support and filling, needs to be prepared. Bio-ink B must have both good biocompatibility and good mechanical properties. The materials for bio-ink B can be synthetic hydrogels and composite hydrogels.

[0087] In the biomimetic tissue construction method, bio-ink B is manifested as follows: In the single-layer bio-printing process, after the bio-array is formed in the printing chamber 52, the printing base 31 moves to the feeding station, the barrel 32 moves to the working position, and the extrusion nozzle of the barrel 32 extends into the printing chamber 52 to suspend and print bio-ink B (i.e., supporting biomaterial) at specific edge positions of the bio-array to form a framework. Then, the photocuring device 34 performs photo-pre-crosslinking on the bio-array to form a single-layer bio-array; or, in the single-layer bio-printing process, after the photocuring device 34 performs photo-pre-crosslinking on the bio-array, the printing base 31 moves to the feeding station, the barrel 32 moves to the working position, and the extrusion nozzle of the barrel 32 extends into the printing chamber 52 to suspend and print supporting biomaterial at specific edge positions of the bio-array to form a framework, thereby obtaining a single-layer bio-array.

[0088] The following two examples further illustrate the 3D printing platform with integrated holographic optical tweezers and the biomimetic tissue construction method proposed in this invention.

[0089] Example 1: Spatial Ordered Arrangement of Different Cell Types

[0090] Preparation of bio-inks: Human pluripotent stem cell-induced cardiomyocytes, endothelial cells, and fibroblasts were digested and shrunk into spherical shapes (10-30 µm in size), then suspended in culture medium. 5% GelMA (methacrylamide gelatin) was added to each, and the mixtures were stirred in a 30°C water bath to prepare bio-inks A-1, A-2, and A-3. Bio-ink B is a hyaluronic acid-sodium alginate composite hydrogel, with a hyaluronic acid to sodium alginate mass ratio of 1:2, designed to improve the biocompatibility and mechanical properties of the ink.

[0091] Organ-on-a-chip fabrication: Using a 5×5cm quartz glass slide as the base, three cell chambers (cell chamber 1, cell chamber 2 and cell chamber 3), one printing chamber and three channels were made using PDMS material. The three cell chambers are connected to the printing chamber through the three channels and are bonded to the base to form an organ-on-a-chip, as shown in Figure 9. The organ-on-a-chip was then sterilized.

[0092] Preparation steps for the 3D printing platform integrating holographic optical tweezers: Bio-inks A-1, A-2, and A-3 are loaded into cartridges 1, 2, and 3 respectively, with the cartridge temperature set to 35 degrees Celsius. Bio-ink B is loaded into cartridge 4, with the temperature set to 25 degrees Celsius. The organ chip is fixed onto the stage. The stage is leveled.

[0093] Material feeding process: The printing base moves to the material feeding station, and cartridge 1 moves to the working position. The extrusion nozzle of cartridge 1 extends into cell chamber 1 and extrudes bio-ink A-1 into cell chamber 1. After bio-ink A-1 is added, cartridge 1 moves to the storage position. Then, the extrusion nozzle of cartridge 2 extends into cell chamber 2 and extrudes bio-ink A-2 into cell chamber 2. After bio-ink A-2 is added, cartridge 2 moves to the storage position. Then, the extrusion nozzle of cartridge 3 extends into cell chamber 3 and extrudes bio-ink A-3 into cell chamber 3. After bio-ink A-3 is added, cartridge 3 moves to the storage position. At this time, the organism is in a disordered suspended state in the ink. Afterward, the printing base moves to the optical tweezers manipulation station.

[0094] Monolayer bioprinting process: The printing base is located at the optical tweezers control station. The holographic optical tweezers module generates a 3×1 optical trap array to capture cardiomyocytes in cell chamber 1. The stage moves horizontally at a speed of 200 μm / s, allowing the cardiomyocytes captured by the optical trap array 3×1 to move through the channel to the printing chamber for arrangement, until a 3×3 square dot matrix cardiomyocyte array is formed in the printing chamber (as shown in Figure 10). The photopolymerization device performs photo-pre-crosslinking on the cardiomyocyte array. After that, the printing base moves to the feeding station, the material cylinder 4 moves to the working position, and the extrusion nozzle of the material cylinder 4 extends into the printing chamber, suspending and printing two parallel frames at the relative edges of the cardiomyocyte array to form a monolayer of cardiomyocytes (as shown in Figure 10).

[0095] Based on a single layer of cardiomyocytes, the single-layer bioprinting process is repeated, stacking single layers of endothelial cells and single layers of fibroblasts vertically upwards (as shown in Figure 11) to obtain a tissue model. The frameworks of adjacent layers are perpendicular to each other, forming a spatial framework structure with high mechanical strength, providing support. A photopolymerization device is used to cross-link the tissue model under light. This achieves the spatially ordered arrangement of cardiomyocytes, endothelial cells, and fibroblasts at the micrometer scale.

[0096] Example 2: Construction of a large-scale cardiac organ module

[0097] Preparation of bio-inks: Atrial cell spheres, left ventricular cell spheres, and right ventricular cell spheres induced by human pluripotent stem cells were suspended in culture medium, and 8% GelMA (methacrylamide gelatin) was added to each sphere. The mixtures were then prepared in a 30°C water bath to produce bio-inks A-1, A-2, and A-3. Bio-ink B is a hyaluronic acid-sodium alginate composite hydrogel mixed with filler cells, wherein the mass ratio of hyaluronic acid to sodium alginate is 1:2. The filler cells are cardiomyocytes, endothelial cells, and fibroblasts induced by human pluripotent stem cells. Its function is to improve the biocompatibility and mechanical properties of the ink and promote the growth of cardiac organoids in the model system.

[0098] Organ-on-a-chip fabrication: Using a 5×5cm quartz glass slide as the base, three cell chambers (cell chamber 1, cell chamber 2 and cell chamber 3), one printing chamber and three channels are made using PDMS material. The three cell chambers are connected to the printing chamber through the three channels and are bonded to the base to form an organ-on-a-chip. The organ-on-a-chip is then sterilized.

[0099] Preparation steps for the 3D printing platform integrating holographic optical tweezers: Bio-inks A-1, A-2, and A-3 are loaded into cartridges 1, 2, and 3 respectively, with the cartridge temperature set to 36 degrees Celsius. Bio-ink B is loaded into cartridge 4, with the temperature set to 25 degrees Celsius. The organ chip is fixed onto the stage. The stage is leveled.

[0100] Material feeding process: The printing base moves to the material feeding station, and cartridge 1 moves to the working position. The extrusion nozzle of cartridge 1 extends into cell chamber 1 and extrudes bio-ink A-1 into cell chamber 1. After bio-ink A-1 is added, cartridge 1 moves to the storage position. Then, the extrusion nozzle of cartridge 2 extends into cell chamber 2 and extrudes bio-ink A-2 into cell chamber 2. After bio-ink A-2 is added, cartridge 2 moves to the storage position. Then, the extrusion nozzle of cartridge 3 extends into cell chamber 3 and extrudes bio-ink A-3 into cell chamber 3. After bio-ink A-3 is added, cartridge 3 moves to the storage position. At this time, the organism is in a disordered suspended state in the ink. Afterward, the printing base moves to the optical tweezers manipulation station.

[0101] The first single-layer bioprinting process: The printing base is located at the optical tweezers control station. The 3×1 optical trap array generated by the holographic optical tweezers module captures atrial cell spheres in cell chamber 1. The stage moves horizontally, allowing the atrial cell spheres captured by the 3×1 optical trap array to move through ramps and channels to the printing chamber. The optical trap array becomes a semi-circle of three dots. After the captured atrial cell spheres are placed in the first position of the printing chamber, the optical tweezers are closed. The cell spheres are relatively large and will not move extensively in the solution after the optical tweezers are closed. The 3×1 optical trap array generated by the holographic optical tweezers module captures left ventricular cell spheres in cell chamber 2. The stage moves horizontally, allowing the left ventricular cell spheres captured by the 3×1 optical trap array to move through ramps and channels to the printing chamber. In the printing chamber, the optical trap array transforms into a three-dot semicircle. After capturing left ventricular cell spheres and placing them in the second position of the printing chamber, the optical tweezers are closed. Since the cell spheres are relatively large, they do not move extensively in the solution after the optical tweezers are closed. The holographic optical tweezers module generates a 3×1 optical trap array to capture right ventricular cell spheres in cell chamber 2. The stage moves horizontally, allowing the right ventricular cell spheres captured by the 3×1 optical trap array to move via a ramp and channel to the printing chamber. The optical trap array transforms into a three-dot semicircle. After capturing right ventricular cell spheres and placing them in the second position of the printing chamber, the optical tweezers are closed. Since the cell spheres are relatively large, they do not move extensively in the solution after the optical tweezers are closed. Thus, a first cell array is formed in the printing chamber (as shown in Figure 12). The printing base moves to the feeding station, the barrel 4 moves to the working position, the extrusion nozzle of the barrel 4 extends into the printing chamber, suspends the printing frame at the edge of the cell array, and prints three support points at three specific positions (as shown in Figure 12). Then the photocuring device performs photo-pre-crosslinking on the cell array to form the first monolayer cell.

[0102] Based on the first monolayer of cells, a second monolayer of cells is vertically stacked and printed upwards (as shown in Figure 12). A 4×1 optical trap array is used in the printing of the second monolayer, and the remaining processes are similar to those of the first monolayer, so they will not be repeated here. Based on the second monolayer, a third monolayer of cells is vertically stacked and printed upwards (as shown in Figure 12). The framework is not printed during the printing of the third monolayer, and the remaining processes are similar to those of the first monolayer, so they will not be repeated here, thus obtaining a tissue model. A photopolymerization device is used to perform overall photopolymerization and cross-linking of the tissue model. In this way, a large-scale biomimetic heart organ module with spatially ordered arrangement from the micrometer to the millimeter scale is constructed.

[0103] In the description of this invention, it should be understood that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of this invention, "a plurality of" means two or more, unless otherwise explicitly specified.

[0104] In this invention, unless otherwise explicitly specified and limited, the terms "installation," "connection," "linking," and "fixing," etc., should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral part; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; they can refer to the internal communication of two components or the interaction between two components. Those skilled in the art can understand the specific meaning of the above terms in this invention according to the specific circumstances.

[0105] In this invention, unless otherwise explicitly specified and limited, "above" or "below" the second feature can mean that the first and second features are in direct contact, or that they are in indirect contact through an intermediate medium. Furthermore, "above," "over," or "on top" the second feature can mean that the first feature is directly above or diagonally above the second feature, or simply indicates that the first feature is at a higher horizontal level than the second feature. "Below," "below," or "beneath" the second feature can mean that the first feature is directly below or diagonally below the second feature, or simply indicates that the first feature is at a lower horizontal level than the second feature.

[0106] In the description of this specification, the terms "one embodiment," "some embodiments," "embodiment," "example," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0107] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make modifications, alterations, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A 3D printing platform integrating holographic optical tweezers, characterized in that, include: A holographic optical tweezers module is used to modulate a laser beam and form an optical trap array in the focal region of the objective lens of the holographic optical tweezers module; The material container (32) is used to store biological materials and can be moved between the storage location and the working location; The printing base (31) is movable between the feeding station and the optical tweezers control station; at the feeding station, the printing base (31) is located below the material cylinder (32), and at the optical tweezers control station, the printing base (31) is located below the objective lens. The stage (33) is mounted on the printing base (31) and can move horizontally; A photocuring device (34) is fixed on a stage (33) and is used to emit light to induce the curing of biological materials; Cell culture plate (5) is fixed on stage (33) and stacked on light curing device (34); cell culture plate (5) has printing chamber (52) and cell chamber (51), and the printing chamber (52) and cell chamber (51) are connected by channel (54); The printing base (31) is located at the feeding station, the barrel (32) moves to the working position, and the extrusion nozzle of the barrel (32) extends into the cell chamber (51) of the cell culture plate (5); the printing base (31) is located at the optical tweezers manipulation station, the optical trap array captures organisms in the cell chamber (51), and the stage (33) moves horizontally so that the organisms captured by the optical trap array move through the channel (54) to the printing chamber (52).

2. The 3D printing platform integrating holographic optical tweezers according to claim 1, characterized in that, The holographic optical tweezers module includes a laser source (11), which is a near-infrared laser source (11), and the objective lens is selected to be 40x, 50x or 100x near-infrared light can pass through the objective lens.

3. The 3D printing platform integrating holographic optical tweezers according to claim 1, characterized in that, It also includes a first power mechanism and a second power mechanism; The first power mechanism is connected to the printing base (31) and drives the printing base (31) to move between the feeding station and the optical tweezers control station; the second power mechanism is installed on the printing base (31) and is connected to the stage (33) and drives the stage (33) to move in the horizontal direction.

4. The 3D printing platform with integrated holographic optical tweezers according to claim 3, characterized in that, The first power mechanism drives the printing base (31) to move along a straight line between the feeding station and the optical tweezers control station, or the first power mechanism drives the printing base (31) to rotate around the axis from the feeding station to the optical tweezers control station.

5. The 3D printing platform integrating holographic optical tweezers according to claim 1, characterized in that, It also includes a barrel moving mechanism and a pneumatic pump; the barrel moving mechanism and the barrel (32) are connected by a transmission, the barrel moving mechanism drives the barrel (32) to move sequentially between the storage position, the middle position and the working position, the barrel moving mechanism drives the barrel (32) to move horizontally from the storage position to the middle position, the barrel moving mechanism drives the barrel (32) to move vertically from the middle position to the working position; the pneumatic pump is connected to the barrel (32), the air pressure generated by the pneumatic pump controls the extrusion of biomaterial from the discharge needle of the barrel (32); The stage (33) can also move in the vertical direction.

6. The 3D printing platform integrating holographic optical tweezers according to claim 1, characterized in that, The light source of the photocuring device (34) is an array of semiconductor light-emitting units laid out in the horizontal direction. The size of the semiconductor light-emitting units is 1~100μm, and each semiconductor light-emitting unit can be driven to emit light independently.

7. The 3D printing platform integrating holographic optical tweezers according to claim 1, characterized in that, In the cell culture plate (5), in the vertical direction, the channel (54) is higher than the bottom surface of the cell chamber (51) and the bottom surface of the printing chamber (52), and the bottom surface of the cell chamber (51) is connected to the channel (54) through the slope (53).

8. A method for constructing biomimetic tissues, characterized in that, The biomimetic tissue construction using the 3D printing platform with integrated holographic optical tweezers as described in any one of claims 1 to 7 includes the following steps: Preparation process: Load the biological materials required for printing into the material cylinder (32), and fix the cell culture plate (5) on the stage (33); Material feeding process: The printing base (31) moves to the feeding station, the material cylinder (32) moves to the working position, the extrusion nozzle of the material cylinder (32) extends into the cell chamber (51) of the cell culture plate (5) and extrudes biological material into the cell chamber (51); after the feeding is completed, the material cylinder (32) moves to the storage position first, and the printing base (31) moves to the optical tweezers control station; Single-layer bioprinting process: The printing base (31) is located at the optical tweezers control station. The optical trap array generated by the holographic optical tweezers module captures the organism in the cell chamber (51). The stage (33) moves horizontally, so that the organism captured by the optical trap array moves through the channel (54) to the printing chamber (52) for arrangement, until a single-layer ordered array of organisms is formed in the printing chamber (52). The photocuring device (34) performs photo-pre-crosslinking on the organism array to form a single-layer micron-scale ordered organism. Repeat the single-layer biological printing process, stack single-layer micron-scale ordered biological bodies in the vertical direction to obtain a tissue model, and use a photocuring device (34) to perform overall cross-linking of the tissue model by light to obtain a biomimetic tissue.

9. The biomimetic tissue construction method according to claim 8, characterized in that, The preparation process also includes: leveling the printing base and stage (33), and positioning the cell culture plate.

10. The biomimetic tissue construction method according to claim 8, characterized in that, In the single-layer bioprinting process, after the bioarray is formed in the printing chamber (52), the printing base (31) moves to the feeding station, the barrel (32) moves to the working position, and the extrusion nozzle of the barrel (32) extends into the printing chamber (52) to suspend and print supporting biomaterials at specific edge positions of the bioarray to form a frame. Then, the photocuring device (34) performs photo-pre-crosslinking on the bioarray to form a single-layer bioarray. Alternatively, in the single-layer bioprinting process, after the photocuring device (34) performs photo-pre-crosslinking on the bioarray, the printing base (31) moves to the feeding station, the barrel (32) moves to the working position, and the extrusion nozzle of the barrel (32) extends into the printing chamber (52) to suspend and print supporting biomaterials at specific edge positions of the bioarray to form a frame, thereby obtaining a single-layer bioarray.

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