WO2021012534A1 - 微流控芯片打印喷嘴和生物3d打印系统 - Google Patents

微流控芯片打印喷嘴和生物3d打印系统 Download PDF

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Publication number
WO2021012534A1
WO2021012534A1 PCT/CN2019/118877 CN2019118877W WO2021012534A1 WO 2021012534 A1 WO2021012534 A1 WO 2021012534A1 CN 2019118877 W CN2019118877 W CN 2019118877W WO 2021012534 A1 WO2021012534 A1 WO 2021012534A1
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Prior art keywords
microfluidic chip
layer
nozzle
flow
flow channel
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PCT/CN2019/118877
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English (en)
French (fr)
Inventor
弥胜利
孙伟
可鑫
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清华大学深圳国际研究生院
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Publication of WO2021012534A1 publication Critical patent/WO2021012534A1/zh

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B33—ADDITIVE MANUFACTURING TECHNOLOGY
    • B33Y—ADDITIVE MANUFACTURING, i.e. MANUFACTURING OF THREE-DIMENSIONAL [3-D] OBJECTS BY ADDITIVE DEPOSITION, ADDITIVE AGGLOMERATION OR ADDITIVE LAYERING, e.g. BY 3-D PRINTING, STEREOLITHOGRAPHY OR SELECTIVE LASER SINTERING
    • B33Y30/00—Apparatus for additive manufacturing; Details thereof or accessories therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M21/00—Bioreactors or fermenters specially adapted for specific uses
    • C12M21/08—Bioreactors or fermenters specially adapted for specific uses for producing artificial tissue or for ex-vivo cultivation of tissue
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/02—Form or structure of the vessel
    • C12M23/16—Microfluidic devices; Capillary tubes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M33/00—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/48—Automatic or computerized control

Definitions

  • the invention relates to a biological 3D printing molding technology, in particular to a microfluidic chip printing nozzle and a biological 3D printing system.
  • 3D printing is a process that uses computer control to deposit materials layer by layer on a platform to build three-dimensional objects.
  • the term 3D printing technology was originally used to describe the process in which the raw material powder is sprayed onto the platform layer by layer under the action of a binder.
  • the commercialization of cost-effective 3D printers has expanded the application of this technology to industries such as architecture, art, automobiles, biomedicine, education, fashion, and toys.
  • 3D printing is widely used in cell research, drug research, cancer research, medical device development, and tissue engineering. Bioprinting combines 3D printing technology, cell biology and material science, combining a printing platform with a device capable of depositing biological ink (biological materials are usually full of active molecules and cells).
  • Bio 3D printing technology can produce a variety of biological materials, such as synthetic or natural polymers as scaffolds, combined with protein-containing serum and extracellular matrix (ECM), and then culture various cells in vitro, including stem cells and somatic cells.
  • biological materials such as synthetic or natural polymers as scaffolds
  • ECM extracellular matrix
  • specific structures, physical and biological properties can be customized to simulate natural tissue functions and provide the microenvironment required for cell growth, proliferation, and controlled differentiation.
  • CAD and CAM enables tissue engineers to use common imaging methods and reconstruction techniques to generate bioprinted tissues with the specific geometric shapes of the organs required by patients.
  • Microfluidic technology usually refers to the technology and science of manipulating micro-volume fluids in micron and below-scale structures. At micro-nano scales, fluids have unique characteristics, interface effects, and thermal conductivity. The outstanding performance of microfluidic preparation fibers shows the great potential of micro-scale separation. The appearance of microfluidic chips further pushes the micro-nano fluidic system to a whole new level. The microfluidic chip can achieve a high degree of integration of various functions. The microfluidic fiber system is a typical representative. Through appropriate chip design, the complete preparation process of samples, sample pretreatment, reaction, multi-component loading and curing can be completed.
  • an integrated chip like this is also called "Lab on a chip.”
  • Lab on a chip an integrated chip like this is also called "Lab on a chip.”
  • the main purpose of the present invention is to overcome the shortcomings of the prior art and provide a microfluidic chip nozzle suitable for printing gradient tissue engineering cornea and a biological 3D printing system suitable for constructing gradient tissue engineering cornea.
  • a nozzle for a microfluidic chip characterized in that it comprises a microfluidic chip and a nozzle with a double-layer structure.
  • the microfluidic chip includes a microfluidic chip base layer and a microfluidic chip upper layer.
  • the microfluidic chip A plurality of flow channels and their inlets, a flow channel of a mixed flow structure connecting the plurality of flow channels and an outlet of the microfluidic chip are formed on the base layer.
  • the flow channel of the mixed flow structure is connected to the outlet of the microfluidic chip, and the micro Microvalves corresponding to the multiple flow channels are formed on the upper layer of the fluid control chip, and the microvalves can be controlled to open or close to control liquids from different flow channels to enter the mixed flow structure flow channel, thereby controlling the flow from the micro The concentration and composition of the mixed solution output from the outlet of the fluidic chip.
  • the nozzle of the double-layer structure includes an inner layer of microneedles and a shell surrounding the outer side of the inner layer of microneedles.
  • the inner layer of microneedles and the microfluidic The control chip outlet is connected, the mixed solution is input into the shell through the inner microneedle, the inner microneedle provides protection for the flow state of the mixed solution after entering the nozzle, and the shell is provided with a cross-linking liquid input port And the nozzle outlet, after the cross-linking liquid passes into the outer shell, it flows along a length of the outer wall of the inner microneedle, wraps the mixed solution flowing out of the inner microneedle in the circumferential direction, and cross-links with the mixed liquid.
  • Gel fibers and use the circumferential fluid focusing effect to control the flow direction of the gel fibers generated by the cross-linking, and flow out from the nozzle outlet.
  • the housing has an inverted cone structure, and the cross-linking liquid input port is arranged at the upper part of the side wall of the housing close to the outlet of the microfluidic chip.
  • the microfluidic chip is arranged vertically, the multiple flow channels include a main flow channel extending downward in a vertical direction and at least one side flow channel connected to the main flow channel, and the flow channel of the mixed flow structure has Two meandering structures extending downward in the vertical direction, and the two meandering structures intersect at least twice during the extension.
  • the mixed flow structure flow channel forms an inverted Y-shaped structure flow channel and a Y-shaped structure flow channel at both ends respectively, and at least two X-shaped structure flow channels are formed in the middle.
  • the inverted Y-shaped structure flow channel, the At least two X-shaped structure flow channels and the Y-shaped structure flow channels are connected in series in sequence.
  • the outlet of the microfluidic chip is arranged as a round hole, and the top of the inner layer microneedle is inserted into the round hole to form a liquid-tight connection.
  • the base layer of the microfluidic chip and the upper layer of the microfluidic chip are all made of soft PDMS mixed with a curing agent, and the ratio of the main agent of the soft PDMS to the curing agent is 10:1.
  • the base layer of the microfluidic chip and the upper layer of the microfluidic chip are bonded together by plasma surface treatment technology.
  • a biological 3D printing system having the microfluidic chip nozzle, a pipe for conveying printing materials to the microfluidic chip nozzle, and for controlling the conveyance to each flow channel on the microfluidic chip nozzle
  • the 3D printing system adopts a circular path printing method in the process of constructing gradient tissue engineering cornea.
  • the gel fibers in the double-layer structure nozzle are deposited on the mold in circles from the inside to the outside. Until the first layer is printed; when the first layer is printed, the nozzle returns to the center of the mold. On the basis of the first layer, the gel fibers are deposited in circles from the inside to the outside in the same way as before.
  • the printed gradient tissue engineered cornea is divided into three layers according to the cell types contained, and the middle layer uses gel fibers containing corneal stromal cells.
  • the inner and outer layers are printed with cell-free gel fibers, and then the inner and outer layers are respectively inoculated with corneal endothelial cells and corneal epithelial cells.
  • the printed tissue engineered cornea is in the radial direction according to whether it contains additional growth factors It is divided into three major circles inside, inside and outside. When the middle circle is printed, the microvalve of the flow channel with high concentration of growth factors in the supply liquid is opened, so that the printed gel fiber contains high concentration of growth factors, and finally achieves different radial directions. Concentration gradient of growth factors, gradient tissue engineering cornea with different cellular components on each layer.
  • a method for printing gradient tissue engineering cornea using the biological 3D printing system is provided.
  • a gradient tissue-engineered cornea printed by the biological 3D printing system The printed gradient tissue-engineered cornea is spatially divided into three layers: inner, middle, and outer according to the cell types contained, and in the radial direction according to whether it contains additional growth factors
  • the upper part is divided into three inner, middle and outer circles.
  • the middle circle is printed with gel fibers containing high concentration of growth factors
  • the inner and outer circles are printed with gel fibers that do not contain high concentration of growth factors
  • the middle layer contains corneal stroma.
  • Cell gel fiber printing, the inner and outer layers are printed with cell-free gel fibers, and then the inner and outer layers are respectively inoculated with corneal endothelial cells and corneal epithelial cells to form different growth factor concentration gradients in the radial direction.
  • Gradient tissue engineered cornea with different cellular components The printed gradient tissue-engineered cornea is spatially divided into three layers: inner, middle, and outer according to the cell types contained, and in the radial direction according to whether it contains additional growth factors
  • the present invention combines biological 3D printing technology and microfluidic technology to provide a microfluidic chip nozzle for biological three-dimensional printing.
  • the structure design of the microfluidic chip nozzle can provide gradient tissue engineering particularly suitable for printing layered structures The fluid shear stress of the cornea.
  • Using the microfluidic chip nozzle of the present invention can accurately print a layered structure of gradient tissue engineering cornea through the control of a microvalve, so that the printed cornea has a three-layer structure of epithelial layer, stromal layer and endothelial layer, and each layer can be inoculated differently. cells.
  • the nozzle of the double-layer structure designed in the present invention includes an inner microneedle and a shell surrounding the inner microneedle.
  • the inner microneedle is connected to the outlet of the microfluidic chip, and the mixed solution enters the nozzle.
  • the latter flow state has a protective effect.
  • the shell is provided with a cross-linking liquid input port and a nozzle outlet. The mixed solution input from the inner microneedle enters the shell.
  • the inner layer of microneedles can flow for a certain length of time, which can completely wrap the mixed solution flowing from the inner layer of microneedles in the circumferential direction, so as to achieve a very good cross-linking phenomenon, and use the circumferential fluid focusing phenomenon to control the condensation caused by cross-linking.
  • the direction of the flow of the glue fiber flows out from the nozzle outlet.
  • Microfluidic chip manufacturing technology makes the manufacture of the nozzle simple, the weight and volume of the nozzle are reduced, and the production cost is reduced;
  • valve on the microfluidic chip can be controlled by the computer to control the flow of each side flow channel in the microfluidic chip, which improves the response speed of the microfluidic chip;
  • the time for each component fluid to enter the mixed flow channel can be controlled by controlling the microvalve switch, so as to control the composition of the fiber prepared by the mixed flow channel;
  • the upper limit of the number of component materials used in the printing process can be determined by controlling the number of component flow channels;
  • multi-component gel fibers without changing nozzles, including: multi-component materials in the same segment of fibers, and multi-segment mono-component fibers in a longer segment of fibers;
  • composition and proportion of the components of the fiber cross-section prepared can be controlled by controlling the number of component runners that flow at the same time;
  • the fiber cross-sectional area can be increased by simultaneously inputting into the mixed flow channel and increasing the flow rate of the component flow channel.
  • the present invention combines microfluidic chip technology and 3D printing technology, makes full use of the continuous and stable characteristics of the microfluidic chip when preparing fibers, and combines the control valve of the present invention to achieve multi-component adjustment in the same hardware Realizing the preparation of fibers of multiple materials at the same outlet can produce gel fibers of different compositions in real time, accurately and flexibly. Taking full advantage of the simple operation, low cost and flexibility of 3D printing technology, the fibers composed of multiple component materials are directly printed on the mold for constructing the tissue engineering cornea, realizing real-time and accurate construction of multi-layer and multi-component gradients Tissue engineering cornea.
  • the tissue engineered cornea is prepared by using the microfluidic chip nozzle of the present invention.
  • the material of each segment of the fiber can be continuously switched according to the composition of the corresponding corneal position during the printing process, and the 3D printing technology can be used for one-time preparation
  • the whole printing process does not need to replace the nozzles, and does not need to be interrupted.
  • Multi-layer multi-component gradient tissue engineering cornea can be completed at one time.
  • the invention is realized based on microfluidic chip technology and 3D printing technology, is simple and easy to implement, has low cost, significant effect, and has excellent advantages and business background.
  • the nozzle of the present invention based on the microfluidic chip technology has excellent characteristics such as simple operation, wide selection of materials, high manufacturing flexibility, and high precision.
  • the nozzle provides an important foundation for realizing the preparation of gradient tissue engineering cornea by the microfluidic chip nozzle And premise.
  • Fig. 1 is a schematic diagram of preparing a gradient tissue engineered cornea by a microfluidic chip nozzle of an embodiment of the present invention.
  • Fig. 2 is an exploded schematic diagram of a microfluidic chip capable of controlling the composition of printing materials and a double-layer structure nozzle that generates gel fibers through cross-linking according to an embodiment of the present invention.
  • Fig. 3 is a schematic diagram of fiber preparation in a method of controlling a multi-component fiber material through a microvalve using a microfluidic chip nozzle of an embodiment of the present invention, the concentration gradient of a single layer growth factor in the radial direction, and the layer-by-layer deposition process of tissue engineering cornea.
  • Fig. 4 is a schematic diagram of the multi-circle structure of multi-layered cells and growth factor concentration of gradient tissue engineering cornea according to an embodiment of the present invention.
  • Fig. 5 is a schematic structural diagram of a microfluidic chip in an embodiment of the present invention.
  • a microfluidic chip nozzle includes a microfluidic chip and a nozzle 2 with a double-layer structure.
  • the microfluidic chip includes a microfluidic chip base layer 10 and The microfluidic chip upper layer 4, the microfluidic chip base layer 10 is formed with a plurality of flow channels 5, 8 and their inlets 6 and a mixed flow structure flow channel 9 connecting the plurality of flow channels 5, 8 and micro flow
  • the outlet of the control chip, the flow channel 9 of the mixed flow structure is connected to the outlet of the microfluidic chip, and the microvalve 7 corresponding to the plurality of flow channels is formed on the upper layer 4 of the microfluidic chip.
  • the microvalve 7 can Controlled opening or closing is used to control the liquids of different flow channels to enter the mixed flow structure flow channel 9 so as to control the concentration and composition of the mixed solution output from the outlet of the microfluidic chip.
  • the double-layer structure nozzle 2 includes internal Layer microneedles 3 and a shell 11 surrounding the inner layer of microneedles 3, the inner layer of microneedles 3 are connected to the outlet of the microfluidic chip, and the mixed solution is input into the shell through the inner layer of microneedles 3 In 11, the inner microneedle 3 has a protective effect on the flow state of the mixed solution after entering the nozzle.
  • the shell 11 is provided with a cross-linking liquid input port and a nozzle outlet.
  • the cross-linking liquid After the cross-linking liquid passes into the shell 11, it passes through Along the length of the inner microneedle 3, the mixed solution flowing out of the inner microneedle 3 can be completely wrapped in the circumferential direction, and the cross-linking liquid input from the cross-linking liquid input port is in the shell 11 Further mixing, so as to achieve a very good cross-linking phenomenon, and use the circumferential fluid focusing phenomenon to control the flow direction of the cross-linked gel fibers, and the gel fibers finally flow out from the nozzle outlet.
  • the structure design of the microfluidic chip nozzle can provide the fluid shear stress of the gradient tissue engineered cornea suitable for printing the layered structure, and the microfluidic chip nozzle of the present invention can accurately print the layered structure through the control of the microvalve Gradient tissue engineering cornea.
  • the housing 11 has an inverted cone structure, and the cross-linking liquid input port is provided on the side wall of the housing 11 near the outlet of the microfluidic chip. Upper part.
  • the microfluidic chip is arranged vertically, and the multiple flow channels include a main flow channel extending downward in the vertical direction and At least one side flow channel connected by the main flow channel, the mixed flow structure flow channel 9 has two meandering structures extending downward in the vertical direction, and the two meandering structures meet at least twice during the extension .
  • the mixed flow structure flow channel 9 respectively forms an inverted Y-shaped structure flow channel and a Y-shaped structure flow channel at the head and tail ends, and at least Two X-shaped structured flow channels, the inverted Y-shaped structured flow channel, the at least two X-shaped structured flow channels, and the Y-shaped structured flow channel are serially connected in sequence.
  • the mixed flow structure according to the preferred embodiment can destroy the laminar flow state of the fluid in the microfluidic chip so that the solution can be fully mixed before flowing out of the flow channel, and the mixing efficiency is high and the effect is good.
  • the mixed fluid is fully mixed and interacts with the cross-linking fluid to produce high-quality gel fibers, which is beneficial to improve the quality of the printed gradient tissue engineering cornea.
  • the outlet of the microfluidic chip is configured as a circular hole, and the top of the inner microneedle 3 is inserted into the circular hole to form a liquid-tight connection.
  • the microfluidic chip base layer 10 and the associated microfluidic chip upper layer 4 are both made of soft PDMS mixed with a curing agent, and the main agent of the soft PDMS and the curing agent The ratio is 10:1.
  • microfluidic chip base layer 10 and the microfluidic chip upper layer 4 are bonded together by plasma surface treatment technology.
  • a biological 3D printing system is provided with the microfluidic chip nozzle, and a pipe (not shown) for conveying printing material to the microfluidic chip nozzle for controlling A pump (not shown in the figure) for delivering the solution flow rate to each flow channel on the nozzle of the microfluidic chip, and a circuit system (not shown in the figure) for controlling the opening and closing of the microvalves on the nozzle of the microfluidic chip show).
  • the 3D printing system uses route planning and adopts a circular path printing method in the process of constructing a gradient tissue engineering cornea.
  • the gel fibers are deposited on the mold in circles from the inside to the outside, until the first layer 12 is printed. After the first layer 12 is printed, the nozzle returns to the center of the mold. Based on the first layer 12, in the same way as before, the gel fibers in the nozzle of the double-layer structure will be circled from the inside to the outside.
  • the ground is deposited on the previous layer, and so on, stacked and printed until the entire corneal model is printed.
  • the printed gradient tissue engineered cornea is divided into inner, middle and outer layers 15, 14, and 16 according to the cell types contained.
  • the middle layer 14 is printed with gel fibers containing corneal stromal cells, and the inner and outer layers 15 , 16 are printed using cell-free gel fibers, and then inoculated with corneal endothelial cells and corneal epithelial cells.
  • the printed tissue engineered cornea is divided into three major circles in the radial direction in the radial direction according to whether it contains additional growth factors. Among them, when printing the large middle circle 17, the control supply contains the flow of high-concentration growth factors. The microvalve of the channel opens, so that the printed fiber contains a high concentration of growth factors. Finally, a gradient tissue engineered cornea with different growth factor concentration gradients 13 in the radial direction and different cellular components on each large layer 14-16 is achieved.
  • a 3D printed gradient tissue engineered cornea the printed gradient tissue engineered cornea is spatially divided into three layers: inner, middle, and outer according to the cell types contained. Whether additional growth factors are included or not is divided into three major circles in the radial direction, the middle circle 17 is printed with gel fibers containing high concentration of growth factors, and the two large circles inside and outside use gels that do not contain high concentrations of growth factors.
  • the middle layer 14 is printed with gel fibers containing corneal stromal cells
  • the inner and outer layers 15, 16 are printed with cell-free gel fibers
  • the inner and outer layers 15, 16 are respectively inoculated with corneal endothelium Cells and corneal epithelial cells form a gradient tissue engineered cornea with different growth factor concentration gradients in the radial direction and different cellular components on different layers.
  • a microfluidic chip nozzle for printing gradient tissue engineering cornea including a microfluidic chip base layer 10, a microfluidic chip upper layer 4, and a double-layer structure nozzle 2.
  • a main flow channel 5 and multiple side flow channels 8 and their inlets 6 are formed, and a mixed flow structure flow channel 9 and an outlet that connect the multiple flow channels and allow different solutions to be fully mixed.
  • the mixed flow structure 9 can destroy the laminar flow state of the fluid in the microfluidic chip so as to allow the solution to be fully mixed before flowing out of the flow channel.
  • Microvalves 7 corresponding to the multiple side flow channels are formed on the upper layer 4 of the microfluidic chip.
  • the microvalves 7 are programmed to control the flow and closure of the flow channels to control the liquids of different concentration components to enter the main flow channels. 5, thereby controlling the concentration and composition of the solution prepared by the mixed flow channel 9 and entering the inner microneedle 3 of the double-layer structure nozzle.
  • the double-layer structure nozzle 2 includes an inner layer of microneedles 3 and a shell 11 that can pass a cross-linking liquid. By continuously passing the cross-linking liquid into the shell 11 and the solution of the inner layer of microneedles 3 for cross-linking, a specific Concentrated gel fibers flow out from the nozzle outlet.
  • the fluid outlets of the base layer 10 of the microfluidic chip and the upper layer 4 of the microfluidic chip are made into circular holes with a 0.5mm puncher, and the inner microneedles 3 of the double-layer nozzle 2 are formed at the outlet. Insert the round hole and seal it with glue.
  • the microfluidic chip base layer 10 and the associated microfluidic chip upper layer 4 are both made of soft PDMS mixed with a hardener.
  • the ratio of the main agent and curing agent of the soft PDMS is 10:1.
  • the base layer 10 of the microfluidic chip and the upper layer 4 of the microfluidic chip are made by molds, and the flow channels are made by soft lithography or nanoimprinting.
  • microfluidic chip base layer 10 and the microfluidic chip upper layer 4 are bonded together by plasma surface treatment technology.
  • the outer diameter of the inner microneedle 3 is 0.85mm and the inner diameter is 0.5mm, and the material is 316 stainless steel.
  • the structure of the double-layer structure nozzle 2 is composed of a side inlet flow channel and a conical shell 11. A liquid that can be cross-linked with the outlet solution of the microfluidic chip is passed into the side inlet flow channel, and cross-linking occurs at the outlet of the inner microneedle to produce gel fibers.
  • a 3D printing system applied to gradient tissue engineering cornea has the microfluidic chip and the double-layer structure nozzle, which is used to infuse the chip and the nozzle with a solution inlet 6.
  • the 3D printing system adopts route planning and adopts a circular path printing method in the process of constructing gradient tissue engineering cornea.
  • the gel fibers in the nozzle of the double-layer structure are drawn from the inside to the outside, circle by circle.
  • the ground is deposited on the mold until the first layer 12 is printed. After the first layer 12 is printed, the nozzle returns to the center of the mold. Based on the first layer 12, in the same way as before, the gel fibers in the nozzle of the double-layer structure will be circled from the inside to the outside.
  • the ground is deposited on the previous layer, and so on, stacked and printed until the entire corneal model is printed.
  • the printed gradient tissue engineered cornea is divided into inner, middle and outer layers 15, 14, and 16 according to the cell types contained.
  • the middle layer 14 is printed with gel fibers containing corneal stromal cells, and the inner and outer layers 15 , 16 are printed using cell-free gel fibers, and then inoculated with corneal endothelial cells and corneal epithelial cells.
  • the printed tissue engineered cornea is divided into three major circles in the radial direction in the radial direction according to whether it contains additional growth factors. Among them, when printing the large middle circle 17, the control supply contains the flow of high-concentration growth factors. The microvalve of the channel opens, so that the printed fiber contains a high concentration of growth factors. Finally, a gradient tissue engineered cornea with different growth factor concentration gradients 13 in the radial direction and different cellular components on each large layer 14-16 is achieved.
  • the gradient tissue engineered cornea 1 can be used to replace the ocular surface of rabbits, perform related animal experiments, and be used for drug screening and toxicity testing.
  • the gradient tissue engineered cornea 1 can be used to study the occurrence and development of corneal-related pathological diseases.

Abstract

一种微流控芯片喷嘴，包括微流控芯片和双层结构的喷嘴，微流控芯片包括微流控芯片基底层和微流控芯片上层，微流控芯片基底层上形成有多条流道和混流结构流道，混流结构流道连接微流控芯片出口，微流控芯片上层上形成有对应于多条流道的微阀，微阀可受控开通或关闭以控制不同流道的液体进入混流结构流道，双层结构的喷嘴包括内层微针头和外壳，内层微针头与微流控芯片出口相连接，外壳上设置有交联液体输入口和喷嘴出口，从内层微针头输入的混合溶液与从交联液体输入口输入的交联液体在外壳内混合，交联产生凝胶纤维并从喷嘴出口流出。本发明的微流控芯片喷嘴结构特别适于打印分层结构的梯度组织工程角膜。

Description

微流控芯片打印喷嘴和生物3D打印系统 技术领域
本发明涉及生物3D打印成型技术，尤其是一种微流控芯片打印喷嘴和生物3D打印系统。
背景技术
3D打印是一种利用计算机控制，在平台上逐层沉积材料来构建三维对象的过程。3D打印技术一词最初用来描述原料粉末在粘合剂的作用下，经由喷墨喷出到平台上层层堆积的过程。近年来，高性价比3D打印机的商业化将这种技术的应用范围扩大到建筑、艺术、汽车、生物医学、教育、时尚、玩具等行业。在生物医学领域，3D打印广泛应用于细胞研究、药物研究、癌症研究、医疗设备开发、和组织工程等方面。生物打印结合了3D打印技术、细胞生物学和材料科学，将打印平台与能够沉积生物墨水的设备(生物材料通常充满了活性分子和细胞)相结合。生物3D打印技术能够制作多种生物材料，例如合成或天然的聚合物作为支架，结合含有蛋白质的血清、细胞外基质(ECM))，进而在体外培养各种细胞，包括干细胞和体细胞等。通过选择适当的生物打印材料和体系结构，可以定制特定的结构、物理和生物特性，以模拟天然组织功能，并提供细胞生长、增殖和受控分化所需的微环境。此外，医学成像、CAD和CAM的并行发展使组织工程师能够使用常见的成像方式和重建技术，以患者所需求器官的特定几何形状生成生物打印组织。
微流控技术，通常是指在微米及以下尺度的结构中操控微小体积流体的技术和科学，在微纳级别尺度下流体具有独特的特征、界面效应及热传导性能。微流控制备纤维的杰出性能展示了微尺度分离的巨大潜力,微流控芯片的出现进一步将微纳流控体系推到了一个全新的高度。微流控芯片可以实现各种功能的高度集成,微流控制纤维系统是一个典型的代表,通过适当的芯片设计可以将样品、样品预处理、反应、多组分装载及固化等完整的制备过程集成在一个芯片上,类似这样的集成芯片也被称为“芯片实验室(Lab on a chip)”。经过二十多年的发展,微流控技术已覆盖化学、物理、 生物、医学、材料科学、光学和微机电系统等众多领域,成为一个重要的交叉学科。
发明内容
本发明的主要目的在于克服现有技术的不足，提供一种适于打印梯度组织工程角膜的微流控芯片喷嘴及适于构建梯度组织工程角膜的生物3D打印系统。
为实现上述目的，本发明采用以下技术方案：
一种微流控芯片喷嘴，其特征在于,包括微流控芯片和双层结构的喷嘴，所述微流控芯片包括微流控芯片基底层和微流控芯片上层，所述微流控芯片基底层上形成有多条流道及其入口和连接所述多条流道的混流结构流道及微流控芯片出口，所述混流结构流道连接所述微流控芯片出口，所述微流控芯片上层上形成有对应于所述多条流道的微阀，所述微阀可受控开通或关闭以控制不同流道的液体进入所述混流结构流道，从而控制从所述微流控芯片出口输出的混合溶液的浓度和成分，所述双层结构的喷嘴包括内层微针头和包围在所述内层微针头的外侧的外壳，所述内层微针头与所述微流控芯片出口相连接，混合溶液通过所述内层微针头输入所述外壳内，所述内层微针头对混合溶液进入喷嘴后的流动状态提供保护，所述外壳上设置有交联液体输入口和喷嘴出口，交联液体在通入外壳后，经过沿着所述内层微针头外壁一段长度的流动，从周向包裹从所述内层微针头流出的混合溶液，与混合液体交联产生凝胶纤维，并利用周向的流体聚焦效应控制交联产生的凝胶纤维的流动方向，从所述喷嘴出口流出。
进一步地：
所述外壳为倒锥形结构，所述交联液体输入口设置在所述外壳的侧壁的靠近所述微流控芯片出口的上部。
所述微流控芯片呈竖直式设置，所述多条流道包括沿竖直方向向下延伸的主流道和与所述主流道相连的至少一条侧流道，所述混流结构流道具有沿竖直方向向下延伸的两条蜿蜒曲折结构，且所述两条蜿蜒曲折结构在延伸途中至少存在两次交汇。
所述混流结构流道在首尾两端分别形成一个倒Y形结构流道和一个Y形结构流道，在中间形成至少两个X形结构流道，所述倒Y形结构流道、所述至少两个X形结构流道和所述Y形结构流道依次串联。
所述微流控芯片出口设置成圆孔，所述内层微针头的顶部插入所述圆孔并形成液密封连接。
所述微流控芯片基底层和所属微流控芯片上层均由混有固化剂的软PDMS制备而成，所述软PDMS的主剂和所述固化剂的比例为10：1。
所述微流控芯片基底层和所述微流控芯片上层通过等离子表面处理技术键合在一起。
一种生物3D打印系统，具有所述的微流控芯片喷嘴，用于向所述微流控芯片喷嘴输送打印材料的管道，用于控制向所述微流控芯片喷嘴上的各流道输送的溶液流速的泵，以及用于控制所述微流控芯片喷嘴上的各微阀开闭的电路系统。
进一步地：
所述3D打印系统通过路线规划，在构建梯度组织工程角膜的过程中采用环形路径的打印方式，将从双层结构喷嘴中的凝胶纤维由内向外，一圈一圈地沉积在模具上，直到打印完成第一层；当第一层打印完毕后，喷嘴重新回到模具圆心处，在第一层的基础上按照和前面相同的方式，由内向外将凝胶纤维一圈一圈地沉积在之前一层上，以此类推，一直到整个角膜模型打印完毕，打印的梯度组织工程角膜按照包含的细胞类型分为内中外三大层，其中中间大层使用包含角膜基质细胞的凝胶纤维打印，内外两大层则使用不含细胞的凝胶纤维打印，之后内外两大层分别接种角膜内皮细胞和角膜上皮细胞，打印的组织工程角膜按照是否包含额外的生长因子又在径向方向上分为里中外三大圈，其中在打印中间大圈时，供应液包含高浓度生长因子的流道的微阀打开，从而打印的凝胶纤维含有高浓度的生长因子，最终实现径向上具有不同生长因子浓度梯度，各大层上具有不同细胞成分的梯度组织工程角膜构建。
一种使用所述的生物3D打印系统打印梯度组织工程角膜的方法。
一种由所述的生物3D打印系统打印的梯度组织工程角膜，打印的梯度组织工程角膜按照包含的细胞类型在空间上分为内中外三大层，按照是否包含额外的生长因子在径向方向上分为里中外三大圈，其中中间大圈使用包含高浓度生长因子的凝胶纤维打印，里外两大圈使用不含高浓度生长因子的凝胶纤维打印,中间大层使用包含角膜基质细胞的凝胶纤维打印，内外两大层使用不含细胞的凝胶纤维打印，之后内外两大层分别接种角膜内皮细胞和角膜上皮细胞，形成在径向上具有不同生长因子浓度梯度，不同大层上具有不同细胞成分的梯度组织工程角膜。
本发明结合生物3D打印技术和微流控技术，提供一种用于生物三维打印的微流控芯片喷嘴，该微流控芯片喷嘴的结构设计能够提供特别适于打 印分层结构的梯度组织工程角膜的流体剪切应力。使用本发明的微流控芯片喷嘴能够通过微阀的控制精确地打印出分层结构的梯度组织工程角膜，使打印的角膜具有上皮层，基质层和内皮层三层结构，每层可接种不同的细胞。
本发明设计的双层结构的喷嘴包括内层微针头和包围在所述内层微针头的外侧的外壳，所述内层微针头与所述微流控芯片出口相连接，对混合溶液进入喷嘴后的流动状态具有保护作用，所述外壳上设置有交联液体输入口和喷嘴出口，从所述内层微针头输入的混合溶液进入外壳内，交联液体在通入外壳后，经过沿着内层微针头一段长度流动的时间，能够完全从周向包裹从内层微针头流出的混合溶液，从而实现效果非常好的交联现象，并利用周向的流体聚焦现象控制通过交联产生凝胶纤维的流动方向，从喷嘴出口流出。
本发明提供的基于微流控技术的微流控芯片喷嘴还具有以下优点：
1)可用于梯度组织工程角膜3D打印，可以通过路径规划，通过一圈一圈从内向外的方式，将凝胶纤维层层堆叠至圆弧形模具上，制备出梯度组织工程角膜；
2)微流控芯片制造技术使得喷嘴的制造变得简单，喷嘴的重量、体积都得到减小，减少制作成本；
3)可以通过计算机控制的控制微流控芯片上的阀门来控制微流控芯片中各侧流道的流通情况，提高了微流控芯片的响应速度；
4)可以通过控制微阀开关控制各组分流体进入混流流道的时间，而实现控制混流流道制备出的纤维的组分；
5)可以通过控制组分流道的数量来决定在打印过程中使用组分材料数量的上限；
6)可以实现在不更换喷嘴的制备出多组分的凝胶纤维，包括:同一段纤维同时存在多组分材料，较长一段纤维同时存在多段单组分材料纤维；
7)可以通过控制同时流动的组分流道的数量来控制制备出的纤维横截面组分的构成和比例；
8)可以通过同时向混流流道中输入并增大组分流道的流速，来实现纤维横截面面积的增大。
本发明结合了微流控芯片技术和3D打印技术，充分利用了微流控芯片制备纤维时，连续稳定的特点，结合了本发明中的控制阀后，通过在同一硬件中多组分调节来实现同一出口制备多种材料的纤维，可以实时、准确、 灵活的制造不同成分的凝胶纤维。充分利用了3D打印技术操作简单、成本低、灵活的特点，将多种组分材料构成的纤维直接打印到构建组织工程角膜的模具上，实现了实时的、准确的搭建多层多成分的梯度组织工程角膜。
根据本发明实施例，利用本发明微流控芯片喷嘴制备组织工程角膜，在打印过程中，可以根据打印过程中对应角膜位置的成分不断切换每一段纤维的材料，可以利用3D打印技术一次性制备而成，整个打印过程无需更换喷嘴，无需中断，可一次性完成多层多成分的梯度组织工程角膜。
本发明基于微流控芯片技术、3D打印技术实现，简单易行、成本低，效果显著，具备极佳的优势和商业背景。
本发明基于微流控芯片技术的喷嘴，具有操作简单、材料选取广泛、较高的制造柔性、精确度高等优秀特性，该喷嘴为实现微流控芯片喷嘴制备梯度组织工程角膜提供了重要的基础和前提。
附图说明
图1是本发明实施例的微流控芯片喷嘴制备梯度组织工程角膜的示意图。
图2是本发明实施例的能够控制打印材料成分的微流控芯片，以及通过交联产生凝胶纤维的双层结构喷嘴的分解示意图。
图3是使用本发明实施例的微流控芯片喷嘴通过微阀控制多成分纤维材料方法中纤维制备，单层生长因子浓度梯度在径向上的改变以及组织工程角膜层层沉积过程的示意图。
图4是本发明实施例的梯度组织工程角膜的多层细胞和生长因子浓度多圈结构示意图。
图5是本发明实施例中的微流控芯片的结构示意图。
具体实施方式
以下对本发明的实施方式作详细说明。应该强调的是，下述说明仅仅是示例性的，而不是为了限制本发明的范围及其应用。
参阅图1至图5，在一种实施例中，一种微流控芯片喷嘴，包括微流控芯片和双层结构的喷嘴2，所述微流控芯片包括微流控芯片基底层10和微流控芯片上层4，所述微流控芯片基底层10上形成有多条流道5、8及其入口6和连接所述多条流道5、8的混流结构流道9及微流控芯片出口，所述混流结构流道9连接所述微流控芯片出口，所述微流控芯片上层4上形成有对应于所述多条流道的微阀7，所述微阀7可受控开通或关闭以控 制不同流道的液体进入所述混流结构流道9，从而控制从所述微流控芯片出口输出的混合溶液的浓度和成分，所述双层结构的喷嘴2包括内层微针头3和包围在所述内层微针头3的外侧的外壳11，所述内层微针头3与所述微流控芯片出口相连接，混合溶液通过所述内层微针头3输入外壳11内，所述内层微针头3对混合溶液进入喷嘴后的流动状态具有保护作用，所述外壳11上设置有交联液体输入口和喷嘴出口，交联液体在通入外壳11后，经过沿着内层微针头3一段长度流动的时间，能够完全从周向包裹从内层微针头3流出的混合溶液，与从所述交联液体输入口输入的交联液体在所述外壳11内进一步混合，从而实现效果非常好的交联现象，并利用周向的流体聚焦现象控制交联产生凝胶纤维的流动方向，所述凝胶纤维最终从所述喷嘴出口流出。该微流控芯片喷嘴的结构设计能够提供适于打印分层结构的梯度组织工程角膜的流体剪切应力，使用本发明的微流控芯片喷嘴能够通过微阀的控制精确地打印出分层结构的梯度组织工程角膜。
参阅图1和图2，在优选的实施例中，所述外壳11为倒锥形结构，所述交联液体输入口设置在所述外壳11的侧壁的靠近所述微流控芯片出口的上部。
参阅图1、图2和图5，在优选的实施例中，所述微流控芯片呈竖直式设置，所述多条流道包括沿竖直方向向下延伸的主流道和与所述主流道相连的至少一条侧流道，所述混流结构流道9具有沿竖直方向向下延伸的两条蜿蜒曲折结构，且所述两条蜿蜒曲折结构在延伸途中至少存在两次交汇。
参阅图1、图2和图5，在更优选的实施例中，所述混流结构流道9在首尾两端分别形成一个倒Y形结构流道和一个Y形结构流道，在中间形成至少两个X形结构流道，所述倒Y形结构流道、所述至少两个X形结构流道和所述Y形结构流道依次串联。
根据优选实施例的混流结构能够破坏微流控芯片内流体的层流状态从而让溶液在流出流道前充分混合，混合的效率高，效果好。混流液充分混合后与交联液作用产生高质量的凝胶纤维，有利于提高打印梯度组织工程角膜的质量。
在优选的实施例中，所述微流控芯片出口设置成圆孔，所述内层微针头3的顶部插入所述圆孔并形成液密封连接。
在优选的实施例中，所述微流控芯片基底层10和所属微流控芯片上层4均由混有固化剂的软PDMS制备而成，所述软PDMS的主剂和所述固化剂 的比例为10：1。
在优选的实施例中，所述微流控芯片基底层10和所述微流控芯片上层4通过等离子表面处理技术键合在一起。
在另一种实施例中，一种生物3D打印系统，具有所述的微流控芯片喷嘴，用于向所述微流控芯片喷嘴输送打印材料的管道(图未示出)，用于控制向所述微流控芯片喷嘴上的各流道输送的溶液流速的泵(图未示出)，以及用于控制所述微流控芯片喷嘴上的各微阀开闭的电路系统(图未示出)。
参见图1、图3、图4，在优选的实施例中，所述3D打印系统通过路线规划，在构建梯度组织工程角膜的过程中采用环形路径的打印方式，将从双层结构喷嘴中的凝胶纤维由内向外，一圈一圈地沉积在模具上，直到打印完成第一层12。当第一层12打印完毕后，喷嘴重新回到模具圆心处，在第一层12的基础上按照和前面相同的方式，由内向外将从双层结构喷嘴中的凝胶纤维一圈一圈地沉积在之前一层上，以此类推，层叠打印，一直到整个角膜模型打印完毕。打印的梯度组织工程角膜按照包含的细胞类型分为内、中、外三大层15、14、16，其中中间大层14使用包含角膜基质细胞的凝胶纤维打印，内、外两大层15、16则使用不含细胞的凝胶纤维打印，之后分别接种角膜内皮细胞和角膜上皮细胞。同时，打印的组织工程角膜按照是否包含额外的生长因子又在径向方向上分为里、中、外三大圈，其中在打印中间大圈17时，控制供应液包含高浓度生长因子的流道的微阀打开，从而打印的纤维含有高浓度的生长因子。最终实现径向上具有不同生长因子浓度梯度13，各大层14-16上具有不同细胞成分的梯度组织工程角膜构建。
如图3和图4所示，在另一种实施例中，一种3D打印的梯度组织工程角膜，打印的梯度组织工程角膜按照包含的细胞类型在空间上分为内中外三大层，按照是否包含额外的生长因子在径向方向上分为里中外三大圈，其中中间大圈17使用包含高浓度生长因子的凝胶纤维打印，里外两大圈使用不含高浓度生长因子的凝胶纤维打印,中间大层14使用包含角膜基质细胞的凝胶纤维打印，内、外两大层15、16使用不含细胞的凝胶纤维打印，之后内外两大层15、16分别接种角膜内皮细胞和角膜上皮细胞，形成在径向上具有不同生长因子浓度梯度，不同大层上具有不同细胞成分的梯度组织工程角膜。
以下结合附图进一步描述本发明具体实施例。
参阅图1至图5，在一种实施例中，一种用于打印梯度组织工程角膜的微流控芯片喷嘴，包括微流控芯片基底层10和微流控芯片上层4以及双 层结构喷嘴2。所述微流控芯片基底层上10形成有一条主流道5和多条侧流道8及其入口6和连接所述多种流道并让不同溶液充分混合的混流结构流道9及出口。所述混流结构9能够破坏微流控芯片内流体的层流状态从而让溶液在流出流道前充分混合。所述微流控芯片上层4上形成有对应于所述多条侧流道的微阀7，所述微阀7通过计算机编程控制流道的流通和闭合进而控制不同浓度成分的液体进入主流道5，从而控制所述混流流道9制备出的对象的溶液浓度和成分并进入双层结构喷嘴的内层微针头3。所述双层结构喷嘴2包括内层微针头3和能够通入交联液体的外壳11，通过向外壳11中持续通入交联液体与内层微针头3的溶液进行交联，从而获得特定浓度成分的凝胶纤维并从喷头出口流出。
优选的实施例中，所述微流控芯片基底层10与微流控芯片上层4的流体出口使用0.5mm的打孔器制作成圆孔，出口处将双层结构喷嘴2的内部微针3插入圆孔并用胶水密封。
优选的实施例中，所述微流控芯片基底层10和所属微流控芯片上层4均由混有硬化剂的软PDMS制备而成。
优选的实施例中，软PDMS的主剂和固化剂的比例为10：1。
优选的实施例中，所述微流控芯片基底层10和所述微流控芯片上层4利用模具制作，通过软光刻方式或纳米压印制作流道。
优选的实施例中，所述微流控芯片基底层10和所属微流控芯片上层4通过等离子表面处理技术键合在一起。
优选的实施例中，所述内层微针头3的外径为0.85mm内径为0.5mm，材料为316不锈钢，所述双层结构喷嘴2结构由一个侧入口流道和圆锥形外壳11组成，所述侧入口流道内通入可以和微流控芯片出口溶液产生交联的液体，并在内层微针头出口处发生交联，产生凝胶纤维。
在另一种实施例中，一种应用于梯度组织工程角膜的3D打印系统，具有所述的微流控芯片和所述的双层结构喷嘴，用于向芯片和喷嘴灌注溶液的流道入口6，用于控制各个流道内溶液流速的数控注射泵，用于控制微阀7开闭的电路系统。
优选的实施例中，所述3D打印系统通过路线规划，在构建梯度组织工程角膜的过程中采用环形路径的打印方式，将从双层结构喷嘴中的凝胶纤维由内向外，一圈一圈地沉积在模具上，直到打印完成第一层12。当第一层12打印完毕后，喷嘴重新回到模具圆心处，在第一层12的基础上按照和前面相同的方式，由内向外将从双层结构喷嘴中的凝胶纤维一圈一圈地 沉积在之前一层上，以此类推，层叠打印，一直到整个角膜模型打印完毕。打印的梯度组织工程角膜按照包含的细胞类型分为内、中、外三大层15、14、16，其中中间大层14使用包含角膜基质细胞的凝胶纤维打印，内、外两大层15、16则使用不含细胞的凝胶纤维打印，之后分别接种角膜内皮细胞和角膜上皮细胞。同时，打印的组织工程角膜按照是否包含额外的生长因子又在径向方向上分为里、中、外三大圈，其中在打印中间大圈17时，控制供应液包含高浓度生长因子的流道的微阀打开，从而打印的纤维含有高浓度的生长因子。最终实现径向上具有不同生长因子浓度梯度13，各大层14-16上具有不同细胞成分的梯度组织工程角膜构建。
优选的实施例中，所述的梯度组织工程角膜1，可以用于替代兔子的眼表，进行相关动物实验，并用于药物的筛选和毒性测试。
优选的实施例中，所述的梯度组织工程角膜1，可以用于角膜相关病理疾病发生及发展过程的研究。
以上内容是结合具体/优选的实施方式对本发明所作的进一步详细说明，不能认定本发明的具体实施只局限于这些说明。对于本发明所属技术领域的普通技术人员来说，在不脱离本发明构思的前提下，其还可以对这些已描述的实施方式做出若干替代或变型，而这些替代或变型方式都应当视为属于本发明的保护范围。

Claims (10)

  1. 一种微流控芯片喷嘴，其特征在于,包括微流控芯片和双层结构的喷嘴，所述微流控芯片包括微流控芯片基底层和微流控芯片上层，所述微流控芯片基底层上形成有多条流道及其入口和连接所述多条流道的混流结构流道及微流控芯片出口，所述混流结构流道连接所述微流控芯片出口，所述微流控芯片上层上形成有对应于所述多条流道的微阀，所述微阀可受控开通或关闭以控制不同流道的液体进入所述混流结构流道，从而控制从所述微流控芯片出口输出的混合溶液的浓度和成分，所述双层结构的喷嘴包括内层微针头和包围在所述内层微针头的外侧的外壳，所述内层微针头与所述微流控芯片出口相连接，混合溶液通过所述内层微针头输入所述外壳内，所述内层微针头对混合溶液进入喷嘴后的流动状态提供保护作用，所述外壳上设置有交联液体输入口和喷嘴出口，交联液体在通入外壳后，经过沿着所述内层微针头外壁一段长度的流动，从周向包裹从所述内层微针头流出的混合溶液，与混合液体交联产生凝胶纤维，并利用周向的流体聚焦效应控制交联产生的凝胶纤维的流动方向，从所述喷嘴出口流出。
  2. 如权利要求1所述的微流控芯片喷嘴，其特征在于,所述外壳为倒锥形结构，所述交联液体输入口设置在所述外壳的侧壁的靠近所述微流控芯片出口的上部。
  3. 如权利要求1或2所述的微流控芯片喷嘴，其特征在于,所述微流控芯片呈竖直式设置，所述多条流道包括沿竖直方向向下延伸的主流道和与所述主流道相连的至少一条侧流道，所述混流结构流道具有沿竖直方向向下延伸的两条蜿蜒曲折结构，且所述两条蜿蜒曲折结构在延伸途中至少存在两次交汇。
  4. 如权利要求3所述的微流控芯片喷嘴，其特征在于,所述混流结构流道在首尾两端分别形成一个倒Y形结构流道和一个Y形结构流道，在中间形成至少两个X形结构流道，所述倒Y形结构流道、所述至少两个X形结构流道和所述Y形结构流道依次串联。
  5. 如权利要求1至4任一项所述的微流控芯片喷嘴，其特征在于,所述微流控芯片出口设置成圆孔，所述内层微针头的顶部插入所述圆孔并形成液密封连接。
  6. 如权利要求1至5任一项所述的微流控芯片喷嘴，其特征在于,所述微流控芯片基底层和所属微流控芯片上层均由混有固化剂的软PDMS制 备而成，所述软PDMS的主剂和所述固化剂的比例为10：1。
  7. 一种生物3D打印系统，其特征在于,具有如权利要求1至6任一项所述的微流控芯片喷嘴，用于向所述微流控芯片喷嘴输送打印材料的管道，用于控制向所述微流控芯片喷嘴上的各流道输送的溶液流速的泵，以及用于控制所述微流控芯片喷嘴上的各微阀开闭的电路系统。
  8. 如权利要求7所述的生物3D打印系统，其特征在于,所述3D打印系统通过路线规划，在构建梯度组织工程角膜的过程中采用环形路径的打印方式，将从双层结构喷嘴中的凝胶纤维由内向外，一圈一圈地沉积在模具上，直到打印完成第一层；当第一层打印完毕后，喷嘴重新回到模具圆心处，在第一层的基础上按照和前面相同的方式，由内向外将凝胶纤维一圈一圈地沉积在之前一层上，以此类推，一直到整个角膜模型打印完毕，打印的梯度组织工程角膜按照包含的细胞类型分为内中外三大层，其中中间大层使用包含角膜基质细胞的凝胶纤维打印，内外两大层则使用不含细胞的凝胶纤维打印，之后内外两大层分别接种角膜内皮细胞和角膜上皮细胞，打印的组织工程角膜按照是否包含额外的生长因子又在径向方向上分为里中外三大圈，其中在打印中间大圈时，供应液包含高浓度生长因子的流道的微阀打开，从而打印的凝胶纤维含有高浓度的生长因子，最终实现径向上具有不同生长因子浓度梯度，各大层上具有不同细胞成分的梯度组织工程角膜构建。
  9. 一种使用如权利要求7或8所述的生物3D打印系统打印梯度组织工程角膜的方法。
  10. 一种由如权利要求7或8所述的生物3D打印系统打印的梯度组织工程角膜，其特征在于,打印的梯度组织工程角膜按照包含的细胞类型在空间上分为内中外三大层，按照是否包含额外的生长因子在径向方向上分为里中外三大圈，其中中间大圈使用包含高浓度生长因子的凝胶纤维打印，里外两大圈使用不含高浓度生长因子的凝胶纤维打印,中间大层使用包含角膜基质细胞的凝胶纤维打印，内外两大层使用不含细胞的凝胶纤维打印，之后内外两大层分别接种角膜内皮细胞和角膜上皮细胞，形成在径向上具有不同生长因子浓度梯度，不同大层上具有不同细胞成分的梯度组织工程角膜。
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