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

Abstract

The invention relates to the technical field of tissue engineering, 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 tweezers module used for forming an optical trap array in an objective lens focal region of the holographic optical tweezers module; the charging barrel is used for storing biological materials and can move between a storage position and a working position; the printing base can move between the feeding station and the optical tweezer control station; the objective table is installed on the printing base and can move in the horizontal direction and the vertical direction; the light curing device is fixed on the objective table and is used for emitting light to induce the biological material to be cured; the cell culture plate is fixed on the objective table and stacked on the photocuring device; the cell culture plate is provided with a printing chamber and a cell chamber, and the printing chamber and the cell chamber are communicated through a channel. A macroscopic ordered three-dimensional structure can be constructed, and ordering and patterning of microscale organisms in the macroscopic three-dimensional structure are also realized.

Description

Technical Field

[0001] 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. Background Art

[0002] Constructing complex and diverse tissue structures and geometric forms, simulating real human tissues (organs), and obtaining bionic tissues (organs) with complete functionality can help reduce rejection, promote cell adhesion, and enhance integration with surrounding tissues. This is very important for the research and application of artificial organ modules in the fields of tissue engineering and regenerative medicine, and is also a challenge currently faced.

[0003] 3D printing uses a computer-assisted layer-by-layer deposition method to position, stack and assemble living cells / cell spheres and biomaterials into special three-dimensional structures to manufacture living tissues and organoids for tissue engineering, regenerative medicine, pharmacokinetics, cancer research and other biological research. 3D printing can construct the spatial structure (shape, size) of relatively macroscopic (centimeter to millimeter) tissues (organs), support the three-dimensional growth of cells, maintain the three-dimensional structure, and has advantages in spatial positioning and maintaining the structure. Biomanufacturing also involves the spatial control of objects at the micron scale and below (cells, cell spheres, microcarriers, etc.), but the current 3D printing technology is slightly insufficient in control at the micron scale due to the limitations of accuracy and resolution, and it is difficult to achieve the spatial positioning of tiny elements inside the macroscopic three-dimensional structure. Therefore, it is necessary to combine other technologies to assist in the precise control of the microscopic level inside the printed structure, so as to achieve precise bionic tissue and organ construction.

[0004] Optical tweezers are a tool that uses the force generated by laser beams to manipulate particles. They can capture and manipulate organisms with high precision, low damage, and non-contact. The basic principle of optical tweezers is that lasers form a three-dimensional light trap by focusing, which exerts a force on objects, namely light radiation pressure. The objects are controlled in the light trap by light radiation pressure, and objects ranging from micrometers to nanometers are manipulated by the movement of the light beam. The magnitude of light radiation pressure is generally in the order of fN to pN. This magnitude of force makes optical tweezers have application prospects in the biomedical field. At present, holographic optical tweezers can be used for cell manipulation, transfer, and sorting of small amounts of cell samples. Optical tweezers technology has the advantages of non-contact elastic control, no mechanical damage, and aseptic operation for biological samples. More importantly, it can directly manipulate micro-nano sized objects, such as stem cells (10-30 microns). However, cell assembly based on optical tweezers technology cannot remain stable for a long time, and the size of manipulation is limited to a small level, and the construction of larger tissues (organs) cannot be achieved. Moreover, capture and manipulation are limited to the plane direction (X, Y axis), and it is difficult to achieve control in the Z axis direction.

[0005] To this end, the effective combination of 3D printing and optical tweezers technology, taking advantage of their strengths and making up for their weaknesses, is of great significance for tissue engineering and organ manufacturing. Summary of the invention

[0006] 1. Technical issues to be resolved

[0007] In view of the above-mentioned shortcomings and deficiencies of the prior art, the present invention provides a 3D printing platform and a bionic tissue construction method integrated with holographic optical tweezers, which can construct a macroscopic three-dimensional structure, and the organism inside the macroscopic three-dimensional structure is ordered and patterned.

[0008] (II) Technical solution

[0009] In order to achieve the above object, the main technical solutions adopted by the present invention include:

[0010] In a first aspect, the present invention provides a 3D printing platform integrated with holographic optical tweezers, comprising:

[0011] A holographic optical tweezers module is used to modulate the laser beam to form a light trap array in the focal area of ​​the objective lens of the holographic optical tweezers module;

[0012] A barrel for storing biological materials and capable of moving between a storage position and a working position;

[0013] The printing base can be moved between the feeding station and the optical tweezers manipulation station; at the feeding station, the printing base is located below the barrel, and at the optical tweezers manipulation station, the printing base is located below the objective lens;

[0014] The stage is mounted on the printing base and can move in the horizontal direction to assist the movement of the light trap;

[0015] A light curing device, fixed on the stage, for emitting light to induce the biomaterial to cure;

[0016] The cell culture plate is fixed on the stage and stacked on the light curing device; the cell culture plate has a printing chamber and a cell chamber, and the printing chamber and the cell chamber are connected through a channel;

[0017] 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 light trap array captures the organism in the cell chamber, and the stage moves in the horizontal direction, so that the organism captured by the light trap array moves to the printing chamber through the channel.

[0018] Optionally, the holographic optical tweezers module includes a laser light source, the laser light source is a near-infrared laser light source, and the objective lens is a 40x, 50x or 100x near-infrared light-transmissive objective lens.

[0019] Optionally, the 3D printing platform with integrated holographic optical tweezers also includes a first power mechanism and a second power mechanism; the first power mechanism is connected to the printing base in a transmission manner to drive the printing base to move between the feeding station and the optical tweezers manipulation station; the second power mechanism is installed on the printing base and is connected to the stage in a transmission manner to drive the stage to move in the horizontal direction.

[0020] Optionally, the first power mechanism drives the printing base to move along a straight line between the loading station and the optical tweezers manipulation station, or the first power mechanism drives the printing base to rotate around an axis from the loading station to the optical tweezers manipulation station.

[0021] Optionally, the 3D printing platform with integrated holographic optical tweezers also includes a barrel moving mechanism and a pneumatic pump; the barrel moving mechanism is transmission-connected to the barrel, the barrel moving mechanism drives the barrel to move in sequence between the storage position, the middle position and the working position, the barrel moving mechanism drives the barrel to move from the storage position to the middle position in the horizontal direction, and the barrel moving mechanism drives the barrel to move from the middle position to the working position in the vertical direction; the pneumatic pump is connected to the barrel, and the air pressure generated by the pneumatic pump controls the extrusion of the biological material from the discharge needle of the barrel; the stage can also move in the vertical direction for focusing the objective lens.

[0022] Optionally, the light source of the light curing device is a semiconductor light emitting unit array laid out in a horizontal direction, the size of the semiconductor light emitting unit is 1 to 100 μm, and each semiconductor light emitting unit can be independently driven to emit light.

[0023] 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 via a slope.

[0024] In a second aspect, the present invention provides a bionic tissue construction method, which uses the above-mentioned 3D printing platform with integrated holographic optical tweezers to construct bionic tissue, comprising the following steps:

[0025] Preparation process: Load the biomaterials required for printing into the barrel and fix the cell culture plate on the stage;

[0026] Feeding process: the printing base moves to the feeding station, the barrel moves to the working position, the extrusion nozzle of the barrel extends into the cell chamber of the cell culture plate, and extrudes the biological material into the cell chamber; after the feeding is completed, the barrel moves to the storage position first, and the printing base moves to the optical tweezers manipulation station;

[0027] Single-layer organism printing process: The printing base is located at the optical tweezers manipulation station, the light trap array generated by the holographic optical tweezers module captures the organism in the cell chamber, and the stage moves in the horizontal direction, so that the organism captured by the light trap array moves through the channel to the printing chamber for arrangement, until a single-layer orderly array of organisms is formed in the printing chamber, and the light curing device pre-crosslinks the organism array by light to form a single-layer micrometer-scale ordered organism;

[0028] The single-layer organism printing process is repeated to stack a single layer of micron-scale ordered organisms in the vertical direction to obtain a tissue model. The light-curing device performs overall cross-linking of the tissue model under light to obtain a bionic tissue.

[0029] Optionally, the preparation process also includes: leveling the printing base and the stage, and positioning the cell culture plate.

[0030] Optionally, in the single-layer organism printing process, after the organism array is formed in the printing chamber, the printing base moves to the feeding station, the barrel moves to the working position, the extrusion nozzle of the barrel extends into the printing chamber, and the supporting biological material is suspended and printed at a specific edge position of the organism array to form a frame, and then the light curing device performs light irradiation pre-crosslinking on the organism array to form a single-layer organism; or,

[0031] In the single-layer organism printing process, after the photocuring device pre-crosslinks the organism array by light irradiation, 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 a specific edge position of the organism array to form a frame and obtain a single-layer organism.

[0032] (III) Beneficial effects

[0033] The beneficial effects of the present invention are:

[0034] The 3D printing platform and bionic tissue construction method of the integrated holographic optical tweezers provided by the present invention are as follows: the printing base moves to the feeding station, the barrel moves to the working position, the extrusion nozzle of the barrel extends into the cell chamber of the cell culture plate, and extrudes the biological material into the cell chamber; the printing base moves to the optical tweezers manipulation station, the light trap array generated by the holographic optical tweezers module captures the organism in the cell chamber, the stage moves in the horizontal direction, so that the organism captured by the light trap array moves to the printing chamber through the channel for arrangement, until a single-layer orderly arranged organism array is formed in the printing chamber, the light curing device performs light irradiation pre-crosslinking on the organism array to form a single-layer organism, and the above process is repeated to stack the single-layer organism in the vertical direction, so as to obtain a bionic tissue with a macroscopic three-dimensional structure. It can be seen that the 3D printing platform and bionic tissue construction method with integrated holographic optical tweezers provided by the present invention combine the advantages of 3D printing in constructing stable three-dimensional structures at the macroscopic (centimeter / millimeter) scale and holographic optical tweezers in assembling cells at the microscopic (micrometer / nanometer) scale, and can construct a macroscopic stable three-dimensional structure, and the organisms inside the macroscopic three-dimensional structure are ordered and patterned, further simulating human tissue, so that the printed tissue (organ) has more complete organ functions. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 It is a structural schematic diagram of a holographic optical tweezers module in a specific implementation manner;

[0036] Figure 2 is a schematic diagram of a light trap lattice in a specific implementation manner;

[0037] Figure 3 It is a structural schematic diagram of a 3D printing platform integrated with holographic optical tweezers in a specific implementation manner;

[0038] Figure 4 It is a structural schematic diagram of a printing base moving along a straight line between a feeding station and an optical tweezers manipulation station in a specific implementation manner;

[0039] Figure 5 It is a structural schematic diagram of the printing base rotating and moving between the feeding station and the optical tweezers manipulation station in a specific implementation manner;

[0040] Figure 6 is a schematic top view of a stage with a light curing device installed in a specific implementation manner;

[0041] Figure 7 is a cross-sectional schematic diagram of a stage equipped with a light curing device in a specific embodiment;

[0042] Figure 8 It is a schematic diagram of the structure of a cell culture plate in a specific embodiment;

[0043] Fig. 9is a top view schematic diagram of the organ chip in Example 1;

[0044] Fig.10 Schematic diagram of a monolayer of cardiomyocytes, a monolayer of endothelial cells and a monolayer of fibroblasts in Example 1;

[0045] Fig.11 It is a schematic diagram of the structure of the organizational model in Example 1;

[0046] Fig.12 This is a schematic diagram of the structures of the first monolayer of cells, the second monolayer of cells, and the third monolayer of cells in Example 2.

[0047] Description of Reference Numerals

[0048] 11: laser light source; 12: diffraction element; 13: lens; 14: dichroic mirror; 15: filter; 16: reflector; 17: objective lens; 18: beam reduction module; 19: illumination light source; 20: camera;

[0049] 31: Printing base; 32: Cylinder; 33: Stage; 34: Light curing device;

[0050] 41: temperature control board; 42: light emitting board;

[0051] 5: Cell culture plate;

[0052] 51: cell chamber; 52: printing chamber; 53: slope; 54: channel. DETAILED DESCRIPTION

[0053] In order to better explain the present invention and facilitate understanding, the present invention is described in detail below in conjunction with the accompanying drawings through specific implementation methods. The "vertical direction" mentioned herein refers to the direction of gravity.

[0054] In order to clearly introduce the 3D printing platform with integrated holographic optical tweezers provided by the present invention, the holographic optical tweezers system and the multi-barrel 32 extrusion 3D printing system are first described below.

[0055] Holographic optical tweezers technology can accurately capture and manipulate multiple tiny objects in three-dimensional space. Its working principle is to use a holographic optical element (diffraction element 12) to modulate the laser beam with the aid of a computer so that the laser beam forms the expected light field distribution in the focal area of ​​the objective lens 17, namely, a light trap array.

[0056] like Figure 1As shown, the holographic optical tweezers system involved in the present invention includes a laser light source 11, a spatial light modulator SLM (including a diffraction element 12), other optical elements (lens 13, dichroic mirror 14, filter 15, reflector 16, objective lens 17, beam reduction module 18), detectors (illumination light source 19, camera 20, etc.) and a printing base 31. Among them, the laser light source 11 is the energy source of the holographic optical tweezers, providing a high-intensity, monochromatic laser beam for forming a light trap array and manipulating particles. SLM is the core component of the holographic optical tweezers, and has the function of converting a single laser beam into a computer-programmed light trap pattern to achieve simultaneous capture and manipulation of multiple particles. Computer programming can design linear, square, semicircular, circular and other horizontal light trap lattices as needed (such as Figure 2 As shown in the figure, and the moving trajectory of the light trap array. SLM is an existing mature optical device, and the specific internal structure and principle will not be described again. Other optical elements, such as lens 13, binocular mirror, filter 15, reflector 16, beam reduction module 18 and other optical elements are necessary components of the entire optical path system, and are used for focusing, filtering, changing the direction of the light beam, etc. Detectors, such as illumination light source 19 and camera 20, are used to monitor and feedback the position and movement state of the organism. In Figure 1 In the figure, the light path indicated by the red arrow is the light path of the laser beam, which is used to form a light trap array, and the light path indicated by the blue arrow is the light path of the LED illumination light, which is used to monitor the position and movement state of the organism.

[0057] The multi-barrel 32-extrusion 3D printing system can achieve orderly three-dimensional spatial accumulation of multiple materials to form a support and fixed structure. Its working principle is to use controllable pressure and precise positioning of the robotic arm to accumulate the bio-ink containing cells, cell spheres, microcarriers and biomaterials into a three-dimensional structure according to the designed model.

[0058] like Figure 3 As shown, the 3D printing platform with integrated holographic optical tweezers provided by the present invention includes: a holographic optical tweezers module, which is used to modulate the laser beam and form a light trap array in the focal area of ​​the objective lens 17 of the holographic optical tweezers module; a barrel 32, which is used to store biological materials and can be moved between a storage position and a working position; a printing base 31, which can be moved between a feeding station and an optical tweezers manipulation station; at the feeding station, the printing base 31 is located below the barrel 32, and at the optical tweezers manipulation station, the printing base 31 is located below the objective lens 17; a stage 33, which is installed on the printing base 31 and can be moved in a horizontal direction; a light curing device 34, which is fixed on the stage 33 and is used to emit light to induce the biological material to solidify; a cell culture plate 5, which is fixed on the stage 33 and stacked on the light curing device 34; the cell culture plate 5 has a printing chamber 52 and a cell chamber 51, and the printing chamber 52 and the cell chamber 51 are connected by a channel 54.

[0059] 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 light trap array captures the organism in the cell chamber 51, and the stage 33 moves in the horizontal direction, so that the organism captured by the light trap array moves to the printing chamber 52 through the channel 54.

[0060] In the 3D printing platform with integrated holographic optical tweezers configured as above, the printing base 31 moves to the feeding station, the barrel 32 moves to the working position, the extrusion nozzle of the barrel 32 extends into the cell chamber 51 of the cell culture plate 5, and extrude the biological material into the cell chamber 51; the printing base 31 moves to the optical tweezers manipulation station, the light trap array generated by the holographic optical tweezers module captures the organism in the cell chamber 51, the stage 33 moves in the horizontal direction, so that the organism captured by the light trap array moves to the printing chamber 52 through the channel 54 for arrangement, until a single-layer orderly arranged organism array is formed in the printing chamber 52, the light curing device 34 performs light pre-crosslinking on the organism array to form a single-layer organism, and the above process is repeated to stack the single-layer organism in the vertical direction, so as to obtain a bionic tissue with a macroscopic three-dimensional structure. It can be seen that the 3D printing platform with integrated holographic optical tweezers provided by the present invention combines the advantages of 3D printing in constructing stable three-dimensional structures at the macroscopic (centimeter / millimeter) scale and holographic optical tweezers in assembling cells at the microscopic (micrometer / nanometer) scale, and can construct macroscopic stable three-dimensional structures, and the organisms inside the macroscopic three-dimensional structures are ordered and patterned, further simulating human tissues, so that the printed tissues (organs) have more complete organ functions.

[0061] It should be noted that the structure of the holographic optical tweezers module is the same as that of the above-mentioned holographic optical tweezers system, which will not be described in detail here.

[0062] Usually, the light source equipped in the holographic optical tweezers system is a 540nm laser light source 11. High-power lasers in the visible light region can damage biological tissues. Preferably, in the present invention, the laser light source 11 of the holographic optical tweezers module is a near-infrared laser light source 11, and the wavelength of the near-infrared laser emitted by the near-infrared laser light source 11 is above 1064nm. Near-infrared lasers in the 1064nm and longer wavelength bands have stronger power (greater than 1W), and stronger-power laser capture and manipulation are more stable, and lasers in this band do not produce strong thermal effects, causing less damage to tissues. Accordingly, each optical element of the holographic optical tweezers module is selected to be compatible with the near-infrared band.

[0063] Preferably, the objective lens 17 of the holographic optical tweezers module is selected to be a 40x, 50x or 100x near-infrared light-transmissive objective lens 17. In this way, the objective lens 17 has a longer working distance, and a larger (vertical) space can be reserved between the objective lens 17 and the printing base 31, which is conducive to accommodating a thicker cell culture plate 5, thereby being able to print a large-sized bionic tissue, and ensuring that the printing base 31 will not collide with the objective lens 17 during movement.

[0064] Preferably, the 3D printing platform with integrated holographic optical tweezers also includes a barrel moving mechanism and a pneumatic pump. The barrel moving mechanism is connected to the barrel 32 by transmission, and the barrel moving mechanism drives the barrel 32 to move between the storage position, the middle position and the working position in sequence. The barrel moving mechanism drives the barrel 32 to move from the storage position to the middle position in the horizontal direction, and the barrel moving mechanism drives the barrel 32 to move from the middle position to the working position in the vertical direction. In this way, the barrel moving mechanism can control the barrel 32 to move to the middle position in the horizontal direction to extend out of the storage position, and then move to the working position in the vertical direction to locate in the cell cavity or printing cavity of the cell culture plate. After the barrel 32 extrude the biomaterial, it first moves to the middle position in the vertical direction, and then moves to the storage position in the horizontal direction to retract to the original position. The pneumatic pump is connected to the barrel 32, and the air pressure generated by the pneumatic pump can extrude the biomaterial from the discharge needle of the barrel 32. In this way, the extrusion of the biomaterial in the barrel 32 is achieved, and the pneumatic pump can be used to control the speed and amount of the biomaterial extruded by the barrel 32 .

[0065] The biological material stored in the cartridge 32 may be fluid biological ink, hydrogel, cells, 3D cell spheroids, micro-nano particles, and the like.

[0066] Furthermore, the barrel 32 has a temperature control function to maintain the properties of the biological material stored in the barrel 32 .

[0067] Generally speaking, the 3D printing platform integrated with holographic optical tweezers includes at least two barrels 32, each of which can store different types of biomaterials. In this way, during the printing process, multiple biomaterials can be arranged and stacked in an orderly manner to construct a bionic tissue with a more complex structure.

[0068] As an example, the barrel moving mechanism is a robotic arm.

[0069] Preferably, the 3D printing platform integrated with 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 by transmission, driving the printing base 31 to move between the feeding station and the optical tweezers manipulation station; the second power mechanism is installed on the printing base 31 and is connected to the stage 33 by transmission, driving the stage 33 to move in the horizontal and vertical directions. The printing base 31 moves between the feeding station and the optical tweezers manipulation station, which is a large-scale movement of centimeters, generally with a moving range of 10 to 20 cm, and the minimum movable distance for each operation is 1 cm. In order to move the organism captured by the light trap array through the channel 54 to the stage 33 of the printing chamber 52, a small range of movement at the nanometer level can be achieved. The minimum movable distance for each operation is 50nm, and the maximum movable distance after multiple operations is 15mm. The moving range is small but the accuracy is higher; therefore, the printing base 31 and the stage 33 are driven to move respectively by two power mechanisms. A low-precision power mechanism can be selected for adaptation to the printing base 31, and a high-precision power mechanism can be selected for adaptation to the stage 33. It is easy to implement and has a simple structure and low cost.

[0070] like Figure 4 As shown, the first power mechanism drives the printing base 31 to move horizontally along a straight line between the feeding station and the optical tweezers manipulation station. Figure 5 As shown, the first power mechanism drives the printing base 31 to rotate horizontally around the axis from the feeding station to the optical tweezers manipulation station. Further, the first power mechanism drives the printing base 31 to rotate horizontally around the axis 90° from the feeding station to the optical tweezers manipulation station.

[0071] Preferably, the stage 33 can also move in the vertical direction. In the printing chamber 52, as the biological material accumulates upward, the working surface (i.e., the accumulation surface of the biological material) will gradually deviate from the focal plane of the objective lens 17, resulting in unclear imaging of the detector of the holographic optical tweezers module, and thus unable to guide the subsequent printing of the biological material according to the imaging; by setting the stage 33 to be movable in the vertical direction, as the biological material accumulates upward, the stage 33 gradually sinks, and the working surface can be continuously stabilized on the focal plane of the objective lens 17.

[0072] Furthermore, the second power mechanism drives the stage 33 to move in the vertical direction.

[0073] like Figure 6 and Figure 7 As shown, in the present invention, the middle of the stage 33 has a cavity, and the light curing device 34 is embedded and installed in the cavity. In this way, the structure is compact and the space utilization rate is high.

[0074] It should be noted that the light curing device can also provide white light illumination, which is used to observe the morphology of cell tissues under bright field.

[0075] Preferably, the light source of the light curing device 34 is an array of semiconductor light emitting units laid out in the horizontal direction, the size of the semiconductor light emitting unit is 1 to 100 μm, and each semiconductor light emitting unit can be driven to emit light independently. In this way, the model in the printing chamber 52 can be photocured more precisely. Furthermore, the light emission of the semiconductor light emitting unit is adjustable within the ultraviolet-visible light range of 250 to 750 nm. As an example, the semiconductor light emitting unit is a micro light emitting diode Micro-LED.

[0076] Preferably, the light curing device 34 includes a temperature control plate 41 and a light emitting plate 42 stacked in sequence from top to bottom, and the cell culture plate 5 is stacked on the temperature control plate 41, and the temperature control plate 41 is made of a transparent material; a temperature sensor and a heating probe are arranged in the temperature control plate 41. In this way, by setting the temperature control plate 41, while ensuring that the light of the light emitting plate 42 passes through the induced light curing, the temperature of the biological material in the cell culture plate 5 can be adjusted, and then the mechanical properties of the biological material can be regulated, which is beneficial to the printing. As an example, the material of the temperature control plate 41 is quartz glass, which has high light transmittance.

[0077] like Figure 8 As shown, 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. Since the light trap captures cells, it can only generate force in the horizontal direction of the cells to restrict the cells. Therefore, the slope 53 is set to connect the bottom surface of the cell chamber 51 and the bottom surface of the channel 54, so that the cells can climb from the cell chamber 51 to the channel 54 and enter the printing chamber 52; the channel 54 is higher than the bottom surface of the cell chamber 51 and the bottom surface of the printing chamber 52, so as to prevent the biological material in the cell chamber 51 from naturally flowing into the printing chamber 52. As an example, the cell culture plate 5 is an organ chip or a cell well plate.

[0078] Furthermore, the organ chip has a printing chamber 52 and at least two cell chambers 51, and the at least two cell chambers 51 are connected to the printing chamber 52 through a channel 54. Providing at least two cell chambers 51 is conducive to the printing of bionic tissues with complex structures.

[0079] The material of the cell culture plate 5 is highly transparent quartz glass, polymethyl methacrylate PMMA or polydimethylsiloxane PDMS.

[0080] Based on the above 3D printing platform with integrated holographic optical tweezers, the present invention also provides a bionic tissue construction method, comprising the following steps:

[0081] Preparation process: put the biological material required for printing into the barrel 32 and install the cell culture plate 5 on the stage 33.

[0082] Feeding process: the printing base 31 moves to the feeding station, the barrel 32 moves to the working position, the extrusion nozzle of the barrel 32 extends into the cell chamber 51 of the cell culture plate 5, and extrude the biological material into the cell chamber 51; after the feeding is completed, the barrel 32 first moves to the storage position, and the printing base 31 then moves to the optical tweezers manipulation station.

[0083] Single-layer organism printing process: the printing base 31 is located at the optical tweezers manipulation station, the light trap array generated by the holographic optical tweezers module captures the organism in the cell chamber 51, and the stage 33 moves in the horizontal direction, so that the organism captured by the light trap array moves to the printing chamber 52 through the channel 54 for arrangement, until a single-layer orderly array of organisms is formed in the printing chamber 52, and the light curing device 34 pre-crosslinks the organism array by light to form a single-layer micrometer-scale ordered organism.

[0084] The single-layer organism printing process is repeated to stack a single layer of micrometer-scale ordered organisms in the vertical direction to obtain a tissue model. The light-curing device 34 performs light-irradiation overall cross-linking on the tissue model to obtain a bionic tissue.

[0085] The preparation process also includes: leveling the printing base and the stage 33, and positioning the cell culture plate.

[0086] In the process of repeating the single-layer biological 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 printing process is continued to be repeated.

[0087] Generally speaking, the bio-ink required for printing is loaded into the barrel 32. Usually, cells, cell spheroids, organoids and microcarriers with a diameter of 15 to 100 microns are used as objects for capture and manipulation and mixed with a bio-medium to form bio-ink A. The bio-medium is a photosensitive material (natural material, synthetic material, composite material) with strong fluidity and high transparency. The fluidity of the bio-medium enables the light trap to stably move the organism in the ink, and the organism can maintain its spatial position for a short time after the light trap disappears. The bio-medium is a photosensitive material and has certain mechanical properties after light pre-curing, and can maintain the array of organisms after the optical tweezers are removed. The bio-medium can be selected from natural hydrogels such as alginate, collagen, gelatin and fibrin.

[0088] Bio-ink A needs to provide a suitable growth environment for organisms. Its physical and chemical properties determine the survival and proliferation of organisms, so the rigidity should not be too high. However, the stacking of organisms in the vertical direction requires rigid materials to provide support, otherwise it will cause excessive squeezing between organisms due to gravity, which is extremely unfavorable for the growth of organisms and the construction of large-scale tissues. For this reason, it is also necessary to prepare bio-ink B for support and filling. Bio-ink B must have certain biocompatibility and good mechanical properties. The material of bio-ink B can be selected from synthetic hydrogels and composite hydrogels.

[0089] The embodiment of biological ink B in the method for constructing bionic tissue is as follows: in the single-layer organism printing process, after the organism 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, and the biological ink B (i.e., the supporting biological material) is suspended and printed at a specific edge position of the organism array to form a frame, and then the photocuring device 34 performs light irradiation pre-crosslinking on the organism array to form a single-layer organism; or, in the single-layer organism printing process, after the photocuring device 34 performs light irradiation pre-crosslinking on the organism 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, and the supporting biological material is suspended and printed at a specific edge position of the organism array to form a frame to obtain a single-layer organism.

[0090] The 3D printing platform integrated with holographic optical tweezers and the bionic tissue construction method proposed in the present invention are further described below through two embodiments.

[0091] Example 1 Spatial Orderly Arrangement of Different Types of Cells

[0092] Preparation of bio-ink: Myocardial cells, endothelial cells and fibroblasts induced by human pluripotent stem cells were digested and shrunk into spheres (size 10-30 μm) and suspended in culture medium. Then 5% GelMA (methacrylated gelatin) was added to the culture medium and mixed in a 30-degree water bath to form bio-inks A-1, A-2 and A-3. Bio-ink B is a hyaluronic acid-sodium alginate composite hydrogel, in which the mass ratio of hyaluronic acid to sodium alginate is 1:2, which improves the biocompatibility and mechanical properties of the ink.

[0093] Preparation of organ chip: A 5×5 cm quartz glass sheet was used as the base, and PDMS material was used to make three cell chambers (cell chamber 1, cell chamber 2, and cell chamber 3), a printing chamber, and three channels. The three cell chambers were connected to the printing chamber through the three channels and bonded to the base to form an organ chip. Fig. 9 As shown, the organ chip is disinfected and sterilized.

[0094] Preparation of the 3D printing platform with integrated holographic optical tweezers: Load bio-inks A-1, A-2, and A-3 into barrels 1, 2, and 3, respectively, and set the barrel temperature to 35 degrees Celsius. Load bio-ink B into barrel 4, and set the temperature to 25 degrees Celsius. Fix the organ chip on the stage. Level the stage.

[0095] Feeding process: the printing base moves to the feeding station, barrel 1 moves to the working position, the extrusion nozzle of barrel 1 extends into cell chamber 1, and extrudes bio-ink A-1 into cell chamber 1. After the addition of bio-ink A-1 is completed, barrel 1 moves to the storage position, and then the extrusion nozzle of barrel 2 extends into cell chamber 2, and extrudes bio-ink A-2 into cell chamber 2. After the addition of bio-ink A-2 is completed, barrel 2 moves to the storage position, and then the extrusion nozzle of barrel 3 extends into cell chamber 3, and extrudes bio-ink A-3 into cell chamber 3. After the addition of bio-ink A-3 is completed, barrel 3 moves to the storage position. At this time, the organism is in a disordered suspended state in the ink. Then the printing base moves to the optical tweezers manipulation station.

[0096] Single-layer bioprinting process: The printing base is located at the optical tweezers manipulation station, and the light trap array 3×1 generated by the holographic optical tweezers module captures the cardiomyocytes in the cell chamber 1. The stage moves at a speed of 200 μm / s in the horizontal direction, so that the cardiomyocytes captured by the light trap array 3×1 move through the channel to the printing chamber for arrangement, until a square dot matrix 3×3 cardiomyocyte array is formed in the printing chamber (such as Fig.10 As shown in the figure, the photocuring device performs light pre-crosslinking on the cardiomyocyte array, and then the printing base moves to the feeding station, the barrel 4 moves to the working position, and the extrusion nozzle of the barrel 4 extends into the printing chamber to suspend and print two parallel frames at the relative edge positions of the cardiomyocyte array to form a single layer of cardiomyocytes (as shown in the figure). Fig.10 shown).

[0097] On the basis of the single-layer cardiomyocytes, the single-layer biological printing process is repeated, and a single layer of endothelial cells and a single layer of fibroblasts are stacked up in the vertical direction (such as Fig.11 As shown in the figure, a tissue model is obtained; the frames of two adjacent layers are perpendicular to each other, forming a spatial frame structure with high mechanical strength, which plays a supporting role; the light curing device performs light cross-linking on the tissue model as a whole, achieving the spatial orderly arrangement of three types of cells, namely, myocardial cells, endothelial cells and fibroblasts at the micrometer scale.

[0098] Example 2 Construction of a large-scale heart organ module

[0099] Preparation of bio-ink: Atrial cell spheres, left ventricular cell spheres and right ventricular cell spheres induced by human pluripotent stem cells were suspended in culture medium, and then 8% GelMA (methacrylated gelatin) was added to the culture medium, and mixed in a 30-degree water bath to form bio-inks A-1, A-2 and A-3. Bio-ink B is a hyaluronic acid-sodium alginate composite hydrogel mixed with filling cells, in which the mass ratio of hyaluronic acid to sodium alginate is 1:2, and the filling cells are cardiomyocytes, endothelial cells and fibroblasts induced by human pluripotent stem cells, which is used to improve the biocompatibility and mechanical properties of the ink and promote the growth of cardiac organoids in the model system.

[0100] Preparation of organ chips: A 5×5 cm quartz glass sheet was used as the base, and PDMS material was used to make three cell chambers (cell chamber 1, cell chamber 2, and cell chamber 3), a printing chamber, and three channels. The three cell chambers were connected to the printing chamber through three channels, respectively, and bonded to the base to form an organ chip, which was then disinfected and sterilized.

[0101] Preparation of the 3D printing platform with integrated holographic optical tweezers: Load bio-inks A-1, A-2, and A-3 into barrels 1, 2, and 3, respectively, and set the barrel temperature to 36 degrees Celsius. Load bio-ink B into barrel 4, and set the temperature to 25 degrees Celsius. Fix the organ chip on the stage. Level the stage.

[0102] Feeding process: the printing base moves to the feeding station, barrel 1 moves to the working position, the extrusion nozzle of barrel 1 extends into cell chamber 1, and extrudes bio-ink A-1 into cell chamber 1. After the addition of bio-ink A-1 is completed, barrel 1 moves to the storage position, and then the extrusion nozzle of barrel 2 extends into cell chamber 2, and extrudes bio-ink A-2 into cell chamber 2. After the addition of bio-ink A-2 is completed, barrel 2 moves to the storage position, and then the extrusion nozzle of barrel 3 extends into cell chamber 3, and extrudes bio-ink A-3 into cell chamber 3. After the addition of bio-ink A-3 is completed, barrel 3 moves to the storage position. At this time, the organism is in a disordered suspended state in the ink. Then the printing base moves to the optical tweezers manipulation station.

[0103] The first single-layer biological printing process: the printing base is located at the optical tweezers manipulation station, the light trap array 3×1 generated by the holographic optical tweezers module captures the atrial cell ball in the cell chamber 1, and the stage moves in the horizontal direction, so that the atrial cell ball captured by the light trap array 3×1 moves to the printing chamber through the slope and the channel, and the light trap array becomes a semicircle of 3 dots. After the captured atrial cell ball is placed in the first position of the printing chamber, the optical tweezers are closed. The cell ball is large and will not move in a large range in the solution after the optical tweezers are closed; the light trap array 3×1 generated by the holographic optical tweezers module captures the left ventricular cell ball in the cell chamber 2, and the stage moves in the horizontal direction, so that the left ventricular cell ball captured by the light trap array 3×1 moves through the slope and the channel. To the printing chamber, the light trap array becomes a semicircle with three dots, the captured left ventricular cell sphere is placed in the second position of the printing chamber and the optical tweezers are turned off. The cell sphere is large and will not move widely in the solution after the optical tweezers are turned off; the light trap array 3×1 generated by the holographic optical tweezers module captures the right ventricular cell sphere in the cell chamber 2, and the stage moves in the horizontal direction, so that the right ventricular cell sphere captured by the light trap array 3×1 moves to the printing chamber through the slope and the channel, and the light trap array becomes a semicircle with three dots, the captured right ventricular cell sphere is placed in the second position of the printing chamber and the optical tweezers are turned off. The cell sphere is large and will not move widely in the solution after the optical tweezers are turned off; in this way, a first cell array (such as Fig.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, the printing frame is suspended toward the edge of the cell array, and three support points are printed at three specific positions (such as Fig.12 As shown), a light-curing device then performs light pre-crosslinking on the cell array to form a first monolayer of cells.

[0104] On the basis of the first monolayer of cells, a second monolayer of cells is printed vertically upwardly (eg Fig.12 As shown in FIG. 1 , a 4×1 light trap array is used in the printing process of the second monolayer of cells. The rest of the process is similar to the printing process of the first monolayer of cells and will not be described here. Based on the second monolayer of cells, a third monolayer of cells is printed in a vertical stack (as shown in FIG. 1 ). Fig.12 As shown in the figure, during the printing process of the third monolayer of cells, the frame is no longer printed. The remaining process is similar to the printing process of the first monolayer of cells and will not be repeated here to obtain a tissue model. The light curing device performs light irradiation and overall cross-linking on the tissue model. In this way, the construction of a bionic large-scale heart organ module with orderly spatial arrangement from micrometer to millimeter scale is achieved.

[0105] In the description of the present invention, it should be understood that the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of the features. In the description of the present invention, the meaning of "plurality" is two or more, unless otherwise clearly and specifically defined.

[0106] In the present invention, unless otherwise clearly specified and limited, the terms "installed", "connected", "connected", "fixed" and the like should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection or an indirect connection through an intermediate medium; it can be the internal connection of two elements or the interaction relationship between two elements. For ordinary technicians in this field, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0107] In the present invention, unless otherwise clearly specified and limited, when a first feature is “on” or “below” a second feature, it may be that the first and second features are in direct contact, or that the first and second features are in indirect contact through an intermediate medium. Moreover, when a first feature is “above”, “above” or “above” a second feature, it may be that the first feature is directly above or obliquely above the second feature, or it may simply mean that the first feature is higher in level than the second feature. When a first feature is “below”, “below” or “below” a second feature, it may be that the first feature is directly below or obliquely below the second feature, or it may simply mean that the first feature is lower in level than the second feature.

[0108] In the description of this specification, the description of the terms "one embodiment", "some embodiments", "embodiment", "example", "specific example" or "some examples" etc. 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 invention. In this specification, the schematic representations of the above terms do 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. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, unless they are contradictory.

[0109] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may alter, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A 3D printing platform integrated with holographic optical tweezers, characterized in that: include: A holographic optical tweezers module is used to modulate the laser beam to form a light trap array in the focal area of ​​the objective lens of the holographic optical tweezers module; A barrel (32) for storing biological materials and capable of moving between a storage position and a working position; The printing base (31) is movable between a feeding station and an optical tweezers manipulation station; at the feeding station, the printing base (31) is located below the barrel (32); at the optical tweezers manipulation station, the printing base (31) is located below the objective lens; A stage (33) is mounted on the printing base (31) and is movable in a horizontal direction; A light curing device (34), fixed on the stage (33), for emitting light to induce the biomaterial to cure; A cell culture plate (5) is fixed on a stage (33) and stacked on a light curing device (34); the cell culture plate (5) comprises a printing chamber (52) and a cell chamber (51), and the printing chamber (52) and the cell chamber (51) are connected via a channel (54); The printing base (31) is located at a feeding station, the barrel (32) moves to a working position, and the extrusion nozzle of the barrel (32) extends into a cell chamber (51) of a cell culture plate (5); the printing base (31) is located at an optical tweezers manipulation station, the optical trap array captures an organism in the cell chamber (51), and the stage (33) moves in a horizontal direction, so that the organism captured by the optical trap array moves to the printing chamber (52) through a channel (54).

2. The 3D printing platform with integrated holographic optical tweezers according to claim 1, characterized in that: The holographic optical tweezers module comprises a laser light source (11), wherein the laser light source (11) is a near-infrared laser light source (11), and the objective lens is a 40-fold, 50-fold or 100-fold near-infrared light-transmissive objective lens.

3. The 3D printing platform with integrated holographic optical tweezers according to claim 1, characterized in that: Also includes a first power mechanism and a second power mechanism; The first power mechanism is in transmission connection with the printing base (31) to drive the printing base (31) to move between the feeding station and the optical tweezers manipulation station; the second power mechanism is installed on the printing base (31) and is in transmission connection with the stage (33) to drive 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 manipulation station, or the first power mechanism drives the printing base (31) to rotate around an axis from the feeding station to the optical tweezers manipulation station.

5. The 3D printing platform with integrated holographic optical tweezers according to claim 1, characterized in that: The device also includes a barrel moving mechanism and a pneumatic pump; the barrel moving mechanism is transmission-connected to the barrel (32); the barrel moving mechanism drives the barrel (32) to move in sequence between a storage position, an intermediate position and a working position; the barrel moving mechanism drives the barrel (32) to move from the storage position to the intermediate position in a horizontal direction; and the barrel moving mechanism drives the barrel (32) to move from the intermediate position to the working position in a vertical direction; the pneumatic pump is in communication with the barrel (32); the air pressure generated by the pneumatic pump controls the biological material to be extruded from the discharge needle of the barrel (32); The loading platform (33) can also move in the vertical direction.

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

7. The 3D printing platform with integrated 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) via the slope (53).

8. A method for constructing a bionic tissue, characterized in that: The bionic tissue construction is performed using the 3D printing platform with integrated holographic optical tweezers as claimed in any one of claims 1 to 7, comprising the following steps: Preparation process: loading the biological material required for printing into the barrel (32), and fixing the cell culture plate (5) on the stage (33); Feeding process: the printing base (31) moves to the feeding station, the barrel (32) moves to the working position, the extrusion nozzle of the barrel (32) extends into the cell chamber (51) of the cell culture plate (5), and extrude the biological material into the cell chamber (51); after the feeding is completed, the barrel (32) first moves to the storage position, and the printing base (31) then moves to the optical tweezers manipulation station; Single-layer organism printing process: the printing base (31) is located at the optical tweezers manipulation station, the light trap array generated by the holographic optical tweezers module captures the organism in the cell chamber (51), the stage (33) moves in the horizontal direction, so that the organism captured by the light trap array moves to the printing chamber (52) through the channel (54) for arrangement, until a single-layer orderly array of organisms is formed in the printing chamber (52), and the light curing device (34) performs light irradiation pre-crosslinking on the organism array to form a single-layer micrometer-scale ordered organism; The single-layer organism printing process is repeated to stack a single layer of micrometer-scale ordered organisms in the vertical direction to obtain a tissue model, and a light curing device (34) performs light irradiation and overall cross-linking on the tissue model to obtain a bionic tissue.

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

10. The bionic tissue construction method according to claim 8, characterized in that: In the single-layer organism printing process, after the organism 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, the extrusion nozzle of the barrel (32) extends into the printing chamber (52), and the supporting biological material is suspended and printed at a specific edge position of the organism array to form a frame, and then the light curing device (34) performs light irradiation pre-crosslinking on the organism array to form a single-layer organism; or, in the single-layer organism printing process, after the light curing device (34) performs light irradiation pre-crosslinking on the organism array, the printing base (31) moves to the feeding station, the barrel (32) moves to the working position, the extrusion nozzle of the barrel (32) extends into the printing chamber (52), and the supporting biological material is suspended and printed at a specific edge position of the organism array to form a frame to obtain a single-layer organism.

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