WO2022104626A1 - 基于微流控技术的多功能器官芯片、制备方法及其应用 - Google Patents

基于微流控技术的多功能器官芯片、制备方法及其应用 Download PDF

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WO2022104626A1
WO2022104626A1 PCT/CN2020/129994 CN2020129994W WO2022104626A1 WO 2022104626 A1 WO2022104626 A1 WO 2022104626A1 CN 2020129994 W CN2020129994 W CN 2020129994W WO 2022104626 A1 WO2022104626 A1 WO 2022104626A1
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cell culture
microchannel
layer
chip
microfluidic technology
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French (fr)
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杨慧
于子桐
郝锐
张翊
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深圳先进技术研究院
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • 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
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/34—Measuring or testing with condition measuring or sensing means, e.g. colony counters
    • 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
    • C12M3/00—Tissue, human, animal or plant cell, or virus culture 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
    • C12M3/00—Tissue, human, animal or plant cell, or virus culture apparatus
    • C12M3/06—Tissue, human, animal or plant cell, or virus culture apparatus with filtration, ultrafiltration, inverse osmosis or dialysis means
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues

Definitions

  • the invention belongs to the fields of microfluidic technology and biotechnology, and relates to a multifunctional organ chip based on the microfluidic technology, a preparation method and applications thereof.
  • the eye is the human visual organ and one of the most important organs in the human senses. It consists of the eyeball and its auxiliary structures. About 80% of the information in the brain is obtained through the eyes. With the improvement of economic level, changes in people's lifestyles and changes in my country's population structure, the spectrum of blinding eye diseases has undergone major changes. Corneal diseases, retinal diseases, refractive errors, glaucoma, and metabolic and age-related non-infectious eye diseases with low vision are the challenges faced by my country's eye health work and the focus of future eye health work.
  • microfluidic technology has been reported to study the effect of corneal epithelial barrier on the metabolism of eye drops [2] , the effect of blinking on the process of dry eye disease [3] , and the effect of blinking shear force on the phenotype of corneal epithelial cells [4] .
  • none of the above models restored the intact human corneal structure, which is crucial for recapitulating the corneal microenvironment and drug metabolism studies in ophthalmic diseases.
  • the invention provides a preparation method and application of a multifunctional organ chip based on microfluidic technology, which can not only stably cultivate human corneal cells in a culture area to form a three-dimensional corneal tissue, but also can perform a check on the integrity of the established corneal barrier. Detection, to solve the problem that in vitro two-dimensional culture and in vivo animal experiments are difficult to correctly reflect the human cornea, and to meet the needs of scientific research and clinical applications for in vitro bionic research models.
  • the technical solution of the present invention to solve the above problem is: a multifunctional organ chip based on microfluidic technology, the special features of which are:
  • the base at least includes two structural layers, the structural layer is provided with a microchannel and a cell culture area communicated with the microchannel, the cell culture areas of the two adjacent structural layers are communicated, and a porous membrane is arranged at the connection.
  • test area is also provided on the above-mentioned structural layer, and the test area is communicated with the cell culture area through a microchannel.
  • microchannel on the above-mentioned structural layer includes at least two ports, one is the inlet and the other is the outlet.
  • the above-mentioned porous membrane is a biocompatible porous membrane such as polycarbonate and polydimethylsiloxane, and the pore diameter of the porous membrane ranges from 0.1 to 10 microns.
  • the porous membrane can also be a silicon wafer, on which a porous array is etched by an etching method.
  • the material of the structure layer is polydimethylsiloxane (PDMS) glass, quartz, silicon wafer, polymethyl methacrylate and other commonly used micro-nano processing materials.
  • PDMS polydimethylsiloxane
  • the thickness of the structure layer is greater than or equal to 1 mm, the thickness of the substrate is about 500 microns, and the depth of the microchannel is greater than or equal to 200 microns.
  • the shape of the above cell culture area is a circle, a rectangle, a triangle or a polygon.
  • the number of the above-mentioned cell culture areas is greater than or equal to one.
  • the number of the above-mentioned structural layers is two layers, which are an upper layer and a lower layer respectively.
  • the microchannel and cell culture area in the upper layer are respectively the first microchannel and the first cell culture area, and the microchannel and cell culture area in the lower layer are respectively the second microchannel, the second cell culture area, and the first microchannel. It includes two ports: an upper layer inlet and an upper layer outlet; the second microchannel includes two ports: a lower layer inlet and a lower layer outlet.
  • the first microchannel includes two ports, one is the upper layer inlet and the other is the upper layer outlet; the second microchannel includes at least two ports, one is the lower layer inlet and the other is the lower layer outlet.
  • cells can be inoculated on the upper and lower surfaces of the porous membrane through the first microchannel of the upper layer and the second microchannel of the lower layer, respectively, and the cells cultured in the first cell culture area of the upper layer and the second cell culture area of the lower layer
  • the cells can exchange substances through the micropores on the porous membrane to form a corneal microenvironment, simulating the human eye organ.
  • the present invention also proposes the above-mentioned preparation method of a multifunctional organ chip based on microfluidic technology, which is special in that it includes the following steps:
  • microchannel for fabricating the structural layer in the above step 1) is fabricated by PDMS over-molding, dry etching, wet etching or nanoimprinting.
  • the mode of chip bonding is oxygen plasma bonding, anodic bonding or fusion bonding.
  • each structural layer specifically includes:
  • the silicon wafer is cleaned and dried, then the surface of the silicon wafer is subjected to hydrophilic treatment, and a layer of photoresist is spin-coated on the surface and pre-baked; after the photoresist is pre-baked, the microchannel mask is used to expose and post Bake; immerse the post-baking silicon wafer substrate in the developer solution for development, and harden the film at a certain temperature for a period of time to harden the photoresist and increase its adhesion to the silicon wafer, and then the microchannel positive mold can be obtained;
  • the PDMS prepolymer and PDMS curing agent are mixed according to a certain mass ratio and stirred evenly, and then the mixed PDMS solution is poured onto the micro-channel male mold, wherein the micro-channel male mold is pretreated with a release agent and baked at a certain temperature. to solidify it;
  • Step 1.3 Turn over the PDMS mold, turn over the cured PDMS, and use a punch to make the inlet and outlet of the microchannel; use the punch again to drill holes in the first cell culture area and the test area to obtain the PDMS structure layer.
  • step 2) is specifically:
  • the bottom surface of the PDMS structure layer and the top surface of the glass substrate are treated with oxygen plasma at the same time, and then the two are aligned and bonded;
  • Step B8 Bonding of adjacent PDMS structural layers:
  • the porous membrane is placed on the top of the cell culture area of the PDMS structure layer below; then the bottom surface of the PDMS structure layer below and the top surface of the PDMS structure layer above are treated with oxygen plasma at the same time, and finally the upper and lower PDMS structure layers are aligned to the bond Then, the alignment and bonding of two adjacent PDMS structural layers are completed in turn, and the fabrication of the microfluidic chip is completed.
  • the above-mentioned porous membrane is a polycarbonate membrane or a silicon wafer; when the porous membrane is a silicon wafer, a porous array is etched on the silicon wafer by an etching method.
  • a method for constructing an organ based on a multifunctional organ chip of microfluidic technology which is special in that it includes the following steps:
  • the structural layer can perfuse the culture medium of each cell.
  • step 1) chip pretreatment includes: sterilizing the chip, then adding extracellular matrix to modify the chip, and washing with a culture medium after air-drying.
  • the present invention also proposes a method for constructing an eye organ chip based on a multifunctional organ chip based on microfluidic technology, which is special in that it includes the following steps:
  • Step C1 pre-processing the multifunctional organ chip based on microfluidic technology according to claim 9;
  • Step C2 3D cell culture:
  • the cell suspension was inoculated into the chip through the lower inlet, the chip was inverted, and then transferred to a carbon dioxide incubator for culture.
  • the corneal endothelial cells adhered to the bottom surface of the porous membrane to grow;
  • the cell suspension is inoculated into the chip through the first cell culture area, and then transferred to a carbon dioxide incubator for culture.
  • the cells adhere to the porous membrane interface and grow into monolayer cells.
  • the liquid is sucked from the first cell culture area to establish a gas-liquid interface.
  • the mixed medium of the two cells is continuously perfused at a certain rate from the lower layer inlet, and the lower layer outlet is stably extracted at the same rate, simulating the physiological microenvironment of the human eye.
  • the above-mentioned multifunctional organ chip based on microfluidic technology is pretreated as follows: after the chip is sterilized, a type 1 collagen modified chip is added, air-dried in an ultra-clean typhoon, and washed with DMEM/F12 medium , soak overnight.
  • a method for constructing a corneal injury model and its processing method based on a multifunctional organ chip based on microfluidic technology which is special in that it includes the following steps:
  • the present invention can construct a three-dimensional cell culture model in vitro, and can more accurately simulate the in vivo microenvironment through precise control of parameters such as flow rate, concentration and composition of biological fluids;
  • the present invention constructs a complete human corneal structure, which can evaluate the integrity of the corneal barrier on the sheet;
  • the cell source of the organ chip of the present invention is not limited to cells derived from human eyes, but can also be primary cells, cancer cells, pluripotent stem cells, totipotent stem cells, unipotent stem cells, etc. derived from other organs, which can continue to proliferate and differentiate in vitro That is, the model constructed by the organ chip is universal and applicable to different human organs, such as brain, lung, heart, liver, kidney, intestine, etc.
  • Fig. 1 is the front view of the microfluidic chip designed by the present invention
  • Fig. 2 is the top view of the microfluidic chip designed by the present invention
  • Fig. 3 is the left side view of the microfluidic chip designed by the present invention.
  • FIG. 4 is a three-dimensional view of a microfluidic chip designed by the present invention.
  • Figure 5 is a flow chart of the fabrication of a microfluidic chip
  • FIG. 6 is a schematic diagram of a microfluidic chip fabrication process
  • Figure 7 is a flow chart of constructing a multifunctional eye organ chip
  • Figure 8 is a flowchart of constructing a disease model
  • FIG. 9 is a physical diagram of a micro-nano fluidic chip
  • Figure 10 is a graph showing the results of hematoxylin-eosin staining of corneal epithelial tissue cultured at the air-liquid interface
  • Fig. 11 is the barrier function of the corneal tissue obtained by eye organ chip culture
  • Figure 12 is a graph showing the results of calcein staining of the eye organ chip, wherein A is the corneal epithelial tissue; B is the corneal endothelial tissue.
  • a multifunctional organ chip based on microfluidic technology characterized in that:
  • the substrate 3 includes at least two structural layers, the structural layer is provided with a microchannel and a cell culture area communicated with the microchannel, the cell culture areas of the two adjacent structural layers are connected, and a porous membrane is provided at the connection. 8.
  • a test area is further provided on the structural layer, and the test area communicates with the cell culture area through a microchannel.
  • the microchannel 4 on the structural layer includes at least two ports, one is the inlet and the other is the outlet.
  • the porous membrane 8 is a polycarbonate membrane, polydimethylsiloxane and other porous membranes with biocompatibility, and the pore size of the porous membrane 8 ranges from 0.1 to 10 microns.
  • the porous membrane can also be a silicon wafer, on which a porous array is etched by an etching method.
  • the cell culture area is a three-dimensional cell culture area.
  • the shapes of the three-dimensional cell culture area and the test area are not limited to circles, but can also be rectangles, triangles, and polygons.
  • the design needs to be adjusted, and the test area can be selected to keep and delete according to the test requirements;
  • the number of three-dimensional cell culture areas is not limited to one, and different numbers can be set according to the throughput requirements of the chip, and the three-dimensional cell culture areas can be separated from each other Collusion can also be connected.
  • parameters such as the length, width, and depth of the microchannel are not limited to a specific size, as long as the cell solution can be injected into the chip.
  • the chip includes two structural layers, the upper layer 1 is the upper layer 1 polydimethylsiloxane (PDMS) with the first microchannel 4 , the first cell culture area 5 and the first test area 9 ,
  • the lower layer porous membrane 8 is a polycarbonate membrane with a microporous structure,
  • the lower layer 2 is a lower layer 2 PDMS with a second microchannel 6, a second cell culture zone and a second test zone 10, and
  • the substrate 3 is a glass-based substrate.
  • the round holes in the upper left corner and the lower right corner of the chip of the present invention are the inlet and outlet of the first microchannel 4, respectively, and the lower left corner and upper right corner of the chip are the inlet and outlet of the second microchannel 6, respectively; the three-dimensional cell culture in the middle of the chip
  • the areas are separated by polycarbonate membranes to form a first cell culture area 5 and a second cell culture area 7 with a diameter of 2-10 mm; the test area in the middle of the chip is interconnected with the cell culture area through microchannels.
  • the thickness of the upper layer 1PDMS and the lower layer 2PDMS is greater than or equal to 1 mm, the thickness of the glass substrate is approximately equal to 500 microns, and the depths of the first microchannel 4 and the second microchannel 6 are greater than or equal to 200 microns;
  • the two microchannels 6 are connected, and the test area is connected with the first cell culture area 5 and the second cell culture area 7 respectively, so that the physiological state of cells or tissues can be detected in real time during the cell culture process.
  • a multifunctional organ chip based on microfluidic technology includes an upper inlet 11 , a lower inlet 13 , an upper outlet 12 , a lower outlet 14 , a first cell culture area 5 and a first testing area 9 .
  • cells can be seeded on the upper and lower surfaces of the porous membrane 8 through the first microchannel 4 of the upper layer 1 and the second microchannel 6 of the lower layer 2, respectively, and the first cell culture area 5 of the upper layer 1 and the lower layer 2
  • the cells cultured in the second cell culture area 7 can achieve material exchange through the micropores on the porous membrane 8 to form a corneal microenvironment, simulating human eye organs.
  • the invention is a multifunctional organ chip based on microfluidic technology, which belongs to the microfluidic chip, and has a unique structure, including a three-dimensional cell culture area and a test area, which can realize the co-culture of multiple cells or multiple tissues, and at the same time It can meet the needs of real-time detection of cell and tissue growth status; at the same time, the structure of the microfluidic chip is scalable, and the number, shape and number of layers of the three-dimensional cell culture area can be designed according to the experimental requirements.
  • a preparation method of a multifunctional organ chip based on microfluidic technology take two structural layers as an example:
  • the chip fabrication process includes three parts: upper-layer 1PDMS fabrication, lower-layer 2PDMS fabrication, and chip bonding, as shown in Figure 5.
  • the fabrication steps of the upper layer 1PDMS include: making the upper layer 1 microchannel positive mold, pouring the upper layer 1PDMS, turning the upper layer 1PDMS mold, and punching the upper layer 1PDMS, as shown in sub-picture S1 in FIG. 5 .
  • the manufacturing steps of the lower layer 2PDMS include: the lower layer 2 microchannel positive mold making, the lower layer 2PDMS pouring, the lower layer 2PDMS mold turning, and the lower layer 2PDMS drilling, as shown in sub-figure S2 in FIG. 5 .
  • Step S1 fabricate the upper layer 1PDMS with the first microchannel 4, the first cell culture area 5 and the first test area 9, this step includes sub-steps B1-B3:
  • Sub-step B1 the first microchannel 4 male mold is made:
  • the silicon wafer is first cleaned and dried, then the surface of the silicon wafer is subjected to hydrophilic treatment, and a layer of photoresist is spin-coated on the surface and pre-baked, as shown in sub-picture a in Figure 6;
  • the microchannel reticle is exposed and post-baked, as shown in sub-picture b in Figure 6; the post-baked silicon wafer substrate is immersed in the developer for development, and the film is hardened at a certain temperature for a period of time to harden the photoresist and increase its contact with the silicon wafer.
  • the adhesion force of the first microchannel 4 can be obtained, as shown in the sub-picture c in Figure 6.
  • Sub-step B2 pouring the upper 1PDMS:
  • the PDMS prepolymer and the PDMS curing agent are mixed according to a certain mass ratio and stirred uniformly, and then the mixed PDMS solution is poured onto the male mold of the first microchannel 4, wherein the male mold of the first microchannel 4 is pretreated with a release agent, and is It is cured by baking at a certain temperature, as shown in sub-picture d in Figure 6.
  • Sub-step B3 the upper layer 1PDMS overturns the mold:
  • the upper layer 1PDMS can be obtained by drilling holes with the first test area 9 , as shown in sub-figure f in FIG. 6 .
  • Step S2 fabricating the lower layer 2PDMS with the second microchannel 6, the second cell culture area 7 and the second test area 10, this step includes sub-steps B4-B6:
  • Sub-step B4 the second microchannel 6 positive mold production:
  • the silicon wafer is first cleaned and dried, then the surface of the silicon wafer is subjected to a hydrophilic treatment, and a layer of photoresist is spin-coated on the surface and pre-baked, as shown in sub-picture g in Figure 6;
  • the microchannel reticle is exposed and post-baked, as shown in sub-image h in Figure 6; the post-baked silicon wafer substrate is immersed in the developer for development, and the film is hardened at a certain temperature for a period of time to harden the photoresist and increase its relationship with the silicon wafer.
  • the adhesion force of the second microchannel 6 can be obtained, as shown in sub-image i in Figure 6.
  • the PDMS prepolymer and the PDMS curing agent are mixed according to a certain mass ratio and stirred uniformly, and then the mixed PDMS solution is poured onto the male mold of the second microchannel 6, wherein the male mold of the second microchannel 6 is pretreated with a release agent, and is It is cured by baking at a certain temperature, as shown in sub-picture j in Figure 6.
  • the lower layer 2PDMS can be obtained by drilling holes in the test area, as shown in sub-picture 1 in FIG. 6 .
  • Step S3 chip bonding, this step includes sub-steps B7-B8:
  • the bottom surface of the lower layer 2PDMS and the top surface of the glass substrate were simultaneously subjected to oxygen plasma treatment, and then the two were aligned and bonded, as shown in sub-image m in Fig. 6 .
  • Sub-step B8 the upper layer 1 PDMS is bonded with the lower layer 2 PDMS:
  • the polycarbonate membrane is aligned and placed on top of the second cell culture area 7 of the lower layer 2PDMS, as shown in sub-picture n in Figure 6; then the bottom surface of the lower layer 2PDMS and the top surface of the upper layer 1PDMS are treated with oxygen plasma at the same time, and finally the two are treated with oxygen plasma.
  • the fabrication of the microfluidic chip is completed by aligning and bonding, as shown in sub-figure o in Figure 6.
  • the multi-functional organ chip fabrication method based on microfluidic technology is simple and diverse.
  • the fabrication method of the microchannel array includes PDMS overturning, dry etching, wet etching, and nanoimprinting. Chip bonding can also be used. Channel oxygen plasma bonding, anodic bonding and fusion bonding methods.
  • the upper layer 1 and the lower layer 2 can be made of materials such as PDMS, silicon wafer, quartz, and glass, and are fabricated by methods such as etching.
  • the structural parameters of the microchannel can be flexibly designed, such as depth, width, length, and number, which can be designed or changed according to cell culture and tissue culture requirements.
  • the PDMS material can be replaced with other materials such as glass substrate or polymethyl methacrylate, and then the microchannel array can be etched at the bottom of the glass substrate and other materials by etching method. , or use the nano-imprint method to make a micro-channel array at the bottom of materials such as polymethyl methacrylate; in the chip fabrication step S3, the chip bonding method is not limited to oxygen plasma bonding, and the anode bond can also be selected according to the chip material selection.
  • the polycarbonate film can be replaced with a material such as a silicon wafer, and then an etching method is used to etch a porous array on the silicon wafer.
  • the chip inlet is not limited to one inlet, and multiple inlets can be designed, and each inlet is respectively supplied with a cell solution, a culture solution, a drug solution, a biomolecule solution, and the like.
  • Human organs can be constructed by using the multi-functional organ chip based on the microfluidic technology of the present invention, and the following is an example of the human cornea to illustrate:
  • a method for simulating human eye physiological microenvironment based on a multifunctional organ chip based on microfluidic technology comprising the following steps:
  • the organ chip fabricated above can simulate the human corneal microenvironment. This section will introduce the construction method and operation steps of the eye organ chip in detail, including chip preprocessing, 3D cell culture, sample collection, and analysis and characterization. The flow chart is shown in the figure. 7 is shown.
  • Substep C1 chip preprocessing:
  • microfluidic chip was sterilized by ultraviolet irradiation for more than or equal to 30 minutes in the ultra-clean bench, and the type 1 collagen modified chip was added, placed at 37 degrees Celsius for more than or equal to 2 hours, and dried in the ultra-clean bench for more than or equal to 1 hour, DMEM/F12 The medium was washed more than or equal to 3 times, soaked overnight, and used for later use.
  • the cell suspension was inoculated into the microfluidic chip that had been modified in step C1 through the lower inlet 13, the chip was inverted, and transferred to a 37 degree Celsius, 5% carbon dioxide incubator for 1 day.
  • the cells are attached to the bottom surface of the porous membrane 8 to grow; after the corneal epithelial cells are digested, the cell suspension is passed through the first cell culture area 5 to inoculate the cells into the microfluidic chip, and then transferred to a 37 degree Celsius, 5% carbon dioxide incubator for cultivation After 1 day, the cells adhered to the interface of the porous membrane 8 and grew into monolayer cells, sucked the liquid from the first cell culture area 5, established an air-liquid interface and continued to culture for 14 days, so that the corneal epithelial cells were differentiated into multi-layer cells.
  • the syringe pump is used for perfusion, and the lower inlet 13 is always perfused with the mixed culture medium of the two cells at a certain rate, while the lower outlet 14 is stably drawn at the same rate, simulating the human eye physiological microenvironment.
  • the porous membrane 8 of the first cell culture zone 5 is taken out and the culture fluid is collected through the lower outlet 14 .
  • Step C4 analyze and characterize:
  • Paraffin sections and frozen sections were performed on the porous membrane 8, and the tissue morphology and physiological function of the three-dimensional culture were observed by means of hematoxylin-eosin staining, immunofluorescence staining, etc., using an inverted fluorescence microscope and a laser confocal microscope.
  • the culture medium can be detected not only by polymerase chain reaction, real-time fluorescent quantitative polynucleotide chain reaction and other methods to detect gene expression levels in physiological and pathological processes, but also by immunocytochemical techniques, Western blotting and other methods. , ELISA and other methods to localize, qualitatively and quantitatively study protein expression, and analyze the changes of key factors and characteristic proteins during culture.
  • the cell source of the organ chip is not limited to cells derived from the human eye, but can also be primary cells, cancer cells, pluripotent stem cells, totipotent stem cells, unipotent stem cells, etc. derived from other organs, which can continue to proliferate and differentiate in vitro.
  • Organ-on-a-chip models are versatile and suitable for different human organs, such as brain, lung, heart, liver, kidney, intestine, etc. Chip pretreatment According to different cell sources, different types of collagen, fibronectin, laminin, basement membrane protein or mucopolysaccharide can be selected as extracellular matrix.
  • Embodiment 4 a multifunctional organ chip based on microfluidic technology to construct a corneal injury model and a method for processing the same, comprising the following steps:
  • Model establishment mainly includes disease model, drug treatment, sample collection, analysis and characterization, and its flow chart is shown in Figure 8.
  • the porous membrane 8 of the eye organ chip was drawn through the first cell culture area 5 to simulate human corneal damage caused by environmental factors or mechanical damage.
  • the extracellular vesicles derived from mesenchymal stem cells are used as a new therapeutic drug to promote the repair of corneal injury.
  • the porous membrane 8 of the first cell culture zone 5 is taken out and the culture fluid is collected through the lower outlet 14 .
  • Paraffin sections and frozen sections were performed on the porous membrane 8, and the tissue morphology and physiological function of the three-dimensional culture were observed by means of hematoxylin-eosin staining, immunofluorescence staining, etc., using an inverted fluorescence microscope and a laser confocal microscope.
  • the culture medium can be detected not only by polymerase chain reaction, real-time fluorescent quantitative polynucleotide chain reaction and other methods to detect gene expression levels in physiological and pathological processes, but also by immunocytochemical techniques, Western blotting and other methods. , ELISA and other methods to localize, qualitatively and quantitatively study protein expression, and analyze the changes of key factors and characteristic proteins during culture.
  • the first cell culture area 5 and the first test area 9 can measure the transepithelial resistance value to evaluate the corneal epithelial barrier function.
  • the multifunctional eye organ chip constructed above is obtained, and relevant characterization is performed according to step C4.
  • the corneal injury model constructed above is obtained, and relevant characterization is performed according to step D4.
  • corneal epithelial cells form multi-layered cells after being cultured on a chip; in Figure 11, corneal epithelial cells are cultured on a chip. With the extension of the culture time, the transepithelial resistance value increases, and the side reflects the multi-layered cells and the It has a barrier function; Figure 12 shows that corneal epithelial cells (A) and corneal endothelial cells (B) are cultured on the chip, and the cell morphology is normal and there are many living cells.
  • the model construction method of the present invention is also suitable for constructing physiological processes involving cell migration, such as embryonic development, angiogenesis, wound healing, immune response, inflammatory response, atherosclerosis, Processes such as cancer metastasis; drugs can be traditional small molecule drugs, macromolecule drugs or new gene therapy, cell therapy and cell-free therapy.
  • cells derived from the human eye can be cultured three-dimensionally to form a nearly physiological three-dimensional structure, and the function of the corneal barrier and the expression of key proteins are investigated.
  • the model can be used to establish eye disease models, drug screening, environmental toxicology evaluation, etc., and provide a technical platform for exploring cell morphology research, molecular mechanism and signaling pathway research of human eye diseases.

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Abstract

一种基于微流控技术的多功能器官芯片、制备方法及其应用。基于微流控技术的多功能器官芯片，包括基底，基底上至少包括两个结构层，结构层上设有微通道（4、6）以及与微通道（4、6）连通的细胞培养区（5、7），相邻两个结构层的细胞培养区（5、7）相连通，连通处设有多孔膜（8）。基于微流控技术的多功能器官芯片不仅能够在细胞培养区（5、7）中稳定培养人角膜细胞，形成三维角膜组织，还可以对建立的角膜屏障的完整性进行检测，解决体外二维培养和体内动物实验难以正确反映人角膜的问题，满足科学研究和临床应用对体外仿生研究模型的需求。

Description

基于微流控技术的多功能器官芯片、制备方法及其应用 技术领域
本发明属于微流控技术及生物技术领域，涉及一种基于微流控技术的多功能器官芯片、制备方法及其应用。
背景技术
眼睛是人的视觉器官，是人类感官中最重要的器官之一，其由眼球及其辅助结构组成，大脑中大约有80％的信息都是通过眼睛获取的。随着经济水平的提升，人民生活方式的改变以及我国人口结构的改变，致盲性眼病疾病谱随之发生了重大改变，主要致盲性眼病已由过去的沙眼传染性眼病转变为以白内障、角膜病、视网膜疾病、屈光不正、青光眼以及弱视为主的代谢性和年龄相关性非传染性眼病，这些是我国眼健康工作面临的挑战和未来眼健康工作的重点。
现阶段，对于眼科疾病的病理生理学和新型治疗药物的研发，主要通过二维细胞培养模型、动物模型和人体组织捐赠来实现。2019年，美国国立卫生研究院(NIH)提出加速构建三维眼类器官和体外微生理***，除了更详细地概括体内人体组织的形态和生理反应外，也有望解决相关组织发展，分子信号传导和病理生理机制等长期科学问题，不仅为研究开辟新途径，更为治疗发展和患者护理提出至关重要的指导 [1]。
近十年，基于微流控技术的器官芯片高速发展，因其微环境与生理环境相近、时间和空间维度上能够提供精确控制、易于实现多组织功能研究等特点，逐步成为生物医学领域新一代仿生研究的重要平台。已有报道应用微流控技术研究角膜上皮屏障作用对眼药水代谢的影响 [2]、眨眼对干眼病进程的影响 [3]、眨眼剪切力对角膜上皮细胞表型的影响 [4]。然而，以上模型均未还原完整的人角膜结构，这对于概括眼科疾病中的角膜微环境和药物代谢研究至关重要。
[1]Wright C B,Becker S M,Low L A,et al.Improved ocular tissue models and eye-on-a-chip technologies will facilitate ophthalmic drug development.Journal of Ocular Pharmacology and Therapeutics,2019,36(1).
[2]Bennet D,Estlack Z,Reid T W,et al.A microengineered human corneal epithelium-on-a-chip for eye drops mass transport evaluation.Lab on a Chip,2018(10).
[3]Seo J,Byun W Y,Alisafaei F,et al.Multiscale reverse engineering of the human ocular surface.Nature Medicine,2019,25(8).
[4]Abdalkader R,Kamei K I.Multi-corneal barrier-on-a-chip to recapitulate eye blinking shear stress forces.Lab on a Chip,2020(20).
发明内容
本发明提出一种基于微流控技术的多功能器官芯片制备方法及其应用，其不仅能够在培养区中稳定培养人角膜细胞，形成三维角膜组织，还可以对建立的角膜屏障的完整性进行检测，解决体外二维培养和体内动物实验难以正确反映人角膜的问题，满足科学研究和临床应用对体外仿生研究模型的需求。
本发明解决上述问题的技术方案是：一种基于微流控技术的多功能器官芯片，其特殊之处在于：
包括基底，基底上至少包括两个结构层，结构层上设有微通道以及与微通道连通的细胞培养区，相邻两个结构层的细胞培养区相连通，连通处设有多孔膜。
进一步地，上述结构层上还设有测试区，所述测试区通过微通道与细胞培养区连通。
进一步地，上述结构层上的微通道至少包括两个端口，一个为入口，一个为出口。
进一步地，上述多孔膜为聚碳酸酯、聚二甲基硅氧烷等具有生物兼容性的多孔膜，且多孔膜的孔径范围为0.1～10微米。所述多孔膜也可以为硅片，所述硅片上通过刻蚀方法刻蚀出多孔阵列。
进一步地，上述结构层的材质为聚二甲基硅氧烷(Polydimethylsiloxane,PDMS)玻璃、石英、硅片以及聚甲基丙烯酸甲酯等常用的微纳加工材料。
进一步地，上述结构层的厚度大于等于1毫米，基底厚度大约等于500微米，微通道的深度大于等于200微米。
进一步地，上述细胞培养区的形状为圆形、矩形、三角形或者多边形。
进一步地，上述细胞培养区的数量为大于等于1个。
进一步地，上述结构层的数量为两层，分别为上层、下层。
所述上层内的微通道和细胞培养区分别为第一微通道、第一细胞培养区，下层内的微通道和细胞培养区分别为第二微通道、第二细胞培养区，第一微通道包括两个端口：上层入口和上层出口；第二微通道包括两个端口：下层入口和下层出口。
第一微通道包括两个端口，一个为上层入口，一个为上层出口；第二微通道至少包括两个端口，一个为下层入口，一个为下层出口。
本发明中，细胞可通过上述上层的第一微通道和下层的第二微通道分别接种到多孔膜的上下两个表面，上层的第一细胞培养区和下层的第二细胞培养区中培养的细胞便可通过多孔膜上的微孔实现物质交换，形成角膜微环境，模拟人眼器官。
另外，本发明还提出上述一种基于微流控技术的多功能器官芯片的制备方法，其特殊之处在于，包括以下步骤：
1)制作各结构层，包括：制作结构层的微通道；再对细胞培养区和测试区进行钻孔，获得结构层；
2)芯片键合：
2.1)将一个结构层与基底键合；
2.2)在已键合的结构层细胞培养区放置多孔膜后，再将相邻两个结构层分别键合。
进一步地，上述步骤1)中制作结构层的微通道采用PDMS翻模、干法刻蚀、湿法刻蚀或纳米压印方法制成。
进一步地，上述步骤2)中，芯片键合的方式为氧等离子体键合、阳极键合 或者熔融键合。
进一步地，上述1)制作各结构层具体包括：
1.1)微通道阳模制作；
1.2)材料浇筑；
1.3)翻模，并制作微通道的入口和出口，再对细胞培养区和测试区进行钻孔，获得结构层。
进一步地，上述步骤1.1)微通道阳模制作：
首先对硅片进行清洗并烘干，然后将硅片表面进行亲水化处理，并在表面旋涂一层光刻胶并前烘；光刻胶前烘后，利用微通道掩模版曝光并后烘；将后烘硅片基底浸入显影液中显影，并在一定温度下坚膜一段时间，使光刻胶硬化并增加其与硅片的粘附力，便可获得微通道阳模；
步骤1.2)PDMS浇筑：
将PDMS预聚物和PDMS固化剂按照一定质量比混合并搅拌均匀，然后将混合PDMS溶液浇筑到微通道阳模上，其中微通道阳模经过脱模剂预处理，并在一定温度下烘烤而使其固化；
步骤1.3)PDMS翻模，翻模固化后的PDMS，并利用打孔器制作微通道的入口和出口；再次利用打孔器对第一细胞培养区和测试区进行钻孔，便可获得PDMS结构层。
进一步地，上述步骤2)具体为：
步骤2.1)PDMS结构层与基底键合：
将PDMS结构层的底面和玻璃基底的顶面同时进行氧等离子体处理，然后将两者对准键合；
步骤B8：相邻的PDMS结构层键合：
将多孔膜放置在下方的PDMS结构层的细胞培养区顶端；然后将下方PDMS结构层的底面和上方的PDMS结构层的顶面同时进行氧等离子体处理，最后将上下两PDMS结构层对准键合，依次完成两相邻PDMS结构层对准键合，便完 成了微流控芯片的制作。
进一步地，上述多孔膜为聚碳酸酯膜或者硅片；当多孔膜为硅片时，所述硅片上通过刻蚀方法刻蚀出多孔阵列。
一种基于微流控技术的多功能器官芯片来构建器官的方法，其特殊之处在于，包括以下步骤：
1)芯片预处理；
2)三维细胞培养：
提供器官组织中每一层细胞的细胞悬液，分别按次序通过微通道将细胞悬液注入各结构层中，放入培养箱中培养，使各层细胞贴附于多孔膜生长，期间各层结构层可灌流各细胞的培养基。
进一步地，所述步骤1)芯片预处理包括：对芯片进行灭菌，再加入细胞外基质修饰芯片，风干后用培养基清洗。
另外，本发明还提出一种基于微流控技术的多功能器官芯片来构建眼器官芯片的方法，其特殊之处在于，包括以下步骤：
步骤C1，将权利要求9所述的基于微流控技术的多功能器官芯片预处理；
步骤C2，三维细胞培养：
角膜内皮细胞消化后，将细胞悬液通过下层入口将细胞接种至芯片中，颠倒芯片，移入二氧化碳培养箱中培养，角膜内皮细胞贴附于多孔膜底面生长；再将角膜上皮细胞消化后，将细胞悬液通过第一细胞培养区将细胞接种至芯片中，移入二氧化碳培养箱中培养，细胞贴附于多孔膜界面生长为单层细胞，从第一细胞培养区吸走液体，建立气液界面继续培养，使得角膜上皮细胞分化为多层细胞，期间从下层入口始终以一定速率持续灌注两种细胞的混合培养基，同时下层出口以相同速率稳定抽取，模拟人眼生理微环境。
进一步地，所述步骤C1，将上述基于微流控技术的多功能器官芯片预处理为：将芯片灭菌后，加入一型胶原蛋白修饰芯片，于超净台风干，DMEM/F12培养基清洗，浸泡过夜。
一种基于微流控技术的多功能器官芯片来构建角膜损伤模型及其处理的方法，其特殊之处在于，包括以下步骤：
步骤D1，疾病模型：
基于上述步骤C1、C2构建的模拟人角膜微环境，
利用移液器枪头，通过第一细胞培养区划过眼器官芯片的多孔膜，模拟由于环境因素或机械损伤造成的人角膜损伤；
步骤D2，药物处理：
将促进角膜损伤修复的治疗药物，通过第一细胞培养区加入，浸没角膜芯片，移入二氧化碳中进行处理；
步骤D3，样品收集：
取出第一细胞培养区的多孔膜并通过下层出口收集培养液；
步骤D4，分析表征
对多孔膜进行切片，观察三维培养的组织形态和生理学功能，对培养液进行检测，检测生理和病理过程中的基因表达水平；对蛋白表达进行定位、定性和定量研究，分析培养期间的关键因子、特征蛋白的变化。
本发明的优点：
1)相较于二维细胞培养，本发明可以在体外构建三维细胞培养模型，并且通过对生物流体的流速、浓度以及成分等参数的精确控制，可以更加准确的模拟体内微环境；
2)本发明利用人源细胞构建的体外仿生模型，回避了动物实验始终存在的伦理问题和种属差异问题；
3)本发明构建完整的人角膜结构，可在片上评价角膜屏障的完整性；
4)构建角膜损伤疾病模型，为研究人眼疾病发展、药物代谢和新型治疗方法提供新型研究平台；
5)本发明器官芯片的细胞来源不局限于人眼来源的细胞，也可以是其他器官来源的原代细胞、癌细胞、多能干细胞、全能干细胞、单能干细胞等，能够 在体外继续增殖分化即可，器官芯片构建的模型具有通用性，适用于不同人体器官，如脑、肺、心脏、肝脏、肾脏、肠道等。
附图说明
图1为本发明设计的微流控芯片的前视图；
图2为本发明设计的微流控芯片的俯视图；
图3为本发明设计的微流控芯片的左视图；
图4为本发明设计的微流控芯片的三维图；
图5为微流控芯片制作流程图；
图6为微流控芯片制作过程示意图；
图7为构建多功能眼器官芯片流程图；
图8为构建疾病模型流程图；
图9为微纳流控芯片实物图；
图10为气液界面培养下的角膜上皮组织的苏木精-伊红染色结果图；
图11为眼器官芯片培养所得的角膜组织的屏障功能；
图12为眼器官芯片的钙黄绿素染色结果图，其中A为角膜上皮组织；B为角膜内皮组织。
其中，1、上层，2、下层，3、基底，4、第一微通道，5、第一细胞培养区，6、第二微通道，7、第二细胞培养区，8、多孔膜，9、第一测试区，10、第二测试区，11、上层入口，12、上层出口，13、下层入口，14、下层出口。
具体实施方式
为使本发明实施方式的目的、技术方案和优点更加清楚，下面将结合本发明实施方式中的附图，对本发明实施方式中的技术方案进行清楚、完整地描述，显然，所描述的实施方式是本发明一部分实施方式，而不是全部的实施方式。基于本发明中的实施方式，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施方式，都属于本发明保护的范围。因此，以下对在附图中提供的本发明的实施方式的详细描述并非旨在限制要求保护的本发明的范围，而是仅仅表示本发明的选定实施方式。
一种基于微流控技术的多功能器官芯片，其特征在于：
包括基底3，基底3上至少包括两个结构层，结构层上设有微通道以及与微通道连通的细胞培养区，相邻两个结构层的细胞培养区相连通，连通处设有多孔膜8。
优选地，结构层上还设有测试区，所述测试区通过微通道与细胞培养区连通。
本发明中，结构层上的微通道4至少包括两个端口，一个为入口，一个为出口。
本发明中，所述多孔膜8为聚碳酸酯膜、聚二甲基硅氧烷等具有生物兼容性的多孔膜，且多孔膜8的孔径范围为0.1～10微米。所述多孔膜也可以为硅片，所述硅片上通过刻蚀方法刻蚀出多孔阵列。
细胞培养区为三维细胞培养区，三维细胞培养区和测试区的形状不限于圆形，也可以是矩形、三角形以及多边形等，三维细胞培养区和测试区的尺寸以根据三维细胞培养或组织培养需求设计进行调整，且测试区可以根据测试需求选择保留和删除；三维细胞培养区的数目不局限于一个，可以根据芯片的通量要求设置不同的数量，且三维细胞培养区相互之间可以分开也可以连接串通。优选地，本发明中，微通道的长度、宽度、深度等参数不限于某一特定尺寸，只要能够将细胞溶液注入到芯片内部即可。
实施例1：
一种基于微流控技术的多功能器官芯片，参见图1-图4以及图9。参见图1，芯片包括两层结构层，上层1是带有第一微通道4、第一细胞培养区5以及第一测试区9的上层1聚二甲基硅氧烷(Polydimethylsiloxane,PDMS)，下层多孔膜8是带有微孔结构的聚碳酸酯膜，下层2是带有第二微通道6、第二细胞培养区以及第二测试区10的下层2PDMS，基底3是玻璃基基底。
参见图2，本发明芯片左上角和右下角圆孔分别为第一微通道4的入口和出口，芯片左下角和右上角分别为第二微通道6的入口和出口；芯片中间的三维细 胞培养区由聚碳酸酯膜隔开，形成直径为2～10毫米的第一细胞培养区5和第二细胞培养区7；芯片中间的测试区通过微通道与细胞培养区相互连接。
参见图3，上层1PDMS和下层2PDMS的厚度大于等于1毫米，玻璃基底厚度大约等于500微米，第一微通道4和第二微通道6的深度大于等于200微米；通过第一微通道4和第二微通道6的连接，测试区分别与第一细胞培养区5和第二细胞培养区7相互连通，使得细胞培养过程中能够满足实时检测细胞或组织的生理状态。
参见图4，一种基于微流控技术的多功能器官芯片包括上层入口11、下层入口13、上层出口12、下层出口14、第一细胞培养区5和第一测试区9。
本实施例中，细胞可通过上述上层1的第一微通道4和下层2的第二微通道6分别接种到多孔膜8的上下两个表面，上层1的第一细胞培养区5和下层2的第二细胞培养区7中培养的细胞便可通过多孔膜8上的微孔实现物质交换，形成角膜微环境，模拟人眼器官。
本发明一种基于微流控技术的多功能器官芯片，属于微流控芯片，其结构独特，包括三维细胞培养区和测试区，可实现多种细胞或多种组织共同培养的同时，还能满足实时检测细胞和组织生长状态的需求；同时，微流控芯片的结构具有可扩展性，三维细胞培养区的数量、形状以及层数可以根据实验需求进行设计。
实施例2：
一种基于微流控技术的多功能器官芯片的制备方法：以两层结构层为例：
芯片制作流程包括上层1PDMS制作、下层2PDMS制作以及芯片键合三部分，如图5所示。上层1PDMS的制作步骤包括：上层1微通道阳模制作、上层1PDMS浇筑、上层1PDMS翻模以及上层1PDMS打孔，见图5中子图S1。下层2PDMS的制作步骤包括：下层2微通道阳模制作、下层2PDMS浇筑、下层2PDMS翻模以及下层2PDMS打孔，见图5中子图S2。而后，首先将下层2PDMS与玻璃基底进行键合，然后将聚碳酸酯膜密封下层2PDMS的第二细胞培养区7，最后将 上层1PDMS与下层2PDMS进行位置对准并键合，便完成了微流控芯片的制作，见图5中子图S3。
步骤S1：制作带有第一微通道4、第一细胞培养区5以及第一测试区9的上层1PDMS，该步骤包含子步骤B1～B3：
子步骤B1，第一微通道4阳模制作：
硅片首先进行清洗并烘干，然后将硅片表面进行亲水化处理，并在表面旋涂一层光刻胶并前烘，见图6中子图a；光刻胶前烘后，利用微通道掩模版曝光并后烘，见图6中子图b；将后烘硅片基底浸入显影液中显影，并在一定温度下坚膜一段时间，使光刻胶硬化并增加其与硅片的粘附力，便可获得第一微通道4阳模，见图6中子图c。
子步骤B2，上层1PDMS浇筑：
PDMS预聚物和PDMS固化剂按照一定质量比混合并搅拌均匀，然后将混合PDMS溶液浇筑到第一微通道4阳模上，其中第一微通道4阳模经过脱模剂预处理，并在一定温度下烘烤而使其固化，见图6中子图d。
子步骤B3，上层1PDMS翻模：
翻模固化后的PDMS，并利用打孔器制作第一微通道4的入口和出口，见图6中子图e；再次利用尺寸为2-10毫米的打孔器对第一细胞培养区5和第一测试区9进行钻孔，便可获得上层1PDMS，见图6中子图f。
步骤S2：制作带有第二微通道6、第二细胞培养区7以及第二测试区10的下层2PDMS，该步骤包含子步骤B4～B6：
子步骤B4，第二微通道6阳模制作：
硅片首先进行清洗并烘干，然后将硅片表面进行亲水化处理，并在表面旋涂一层光刻胶并前烘，见图6中子图g；光刻胶前烘后，利用微通道掩模版曝光并后烘，见图6中子图h；将后烘硅片基底浸入显影液中显影，并在一定温度下坚膜一段时间，使光刻胶硬化并增加其与硅片的粘附力，便可获得第二微通道6阳模，见图6中子图i。
子步骤B5，下层2PDMS浇筑：
PDMS预聚物和PDMS固化剂按照一定质量比混合并搅拌均匀，然后将混合PDMS溶液浇筑到第二微通道6阳模上，其中第二微通道6阳模经过脱模剂预处理，并在一定温度下烘烤而使其固化，见图6中子图j。
子步骤B6，下层2PDMS翻模：
翻模固化后的PDMS，并利用打孔器制作第二微通道6的入口和出口，见图6中子图k；再次利用尺寸为2-10毫米的打孔器对第二细胞培养区和测试区进行钻孔，便可获得下层2PDMS，见图6中子图l。
步骤S3，芯片键合，该步骤包含子步骤B7～B8：
子步骤B7，下层2PDMS与玻璃基底键合：
将下层2PDMS的底面和玻璃基底的顶面同时进行氧等离子体处理，然后将两者对准键合，见图6中子图m。
子步骤B8，上层1PDMS与下层2PDMS键合：
将聚碳酸酯膜对准放置在下层2PDMS的第二细胞培养区7顶端，见图6中子图n；然后将下层2PDMS的底面和上层1PDMS的顶面同时进行氧等离子体处理，最后将两者对准键合便完成了微流控芯片的制作，见图6中子图o。
本发明中，基于微流控技术的多功能器官芯片制作方法简单多样，微通道阵列制作方法包括PDMS翻模、干法刻蚀、湿法刻蚀以及纳米压印等方法，芯片键合也可通道氧等离子体键合、阳极键合以及熔融键合等方法。
本发明中的上层1、下层2可以采用PDMS、硅片、石英和玻璃等材料，并利用刻蚀方法等方法进行制作。微通道的结构参数可以灵活设计，比如深度、宽度、长度、数量可以根据细胞培养和组织培养要求进行设计或更改。
芯片制作步骤S1和S2制作上层1PDMS和下层2PDMS时，可以将PDMS材料更换为玻璃基底或聚甲基丙烯酸甲酯等其他材料，然后利用刻蚀方法在玻璃基底等材料底部刻蚀出微通道阵列，或利用纳米压印方法在聚甲基丙烯酸甲酯等材料底部制作出微通道阵列；芯片制作步骤S3中，芯片键合方式不局限于氧等 离子体键合，也可根据芯片选材选择阳极键合以及熔融键合等其他键合方式；芯片制作步骤S3中，可以将聚碳酸酯膜更换为硅片等材料，再利用刻蚀方法在硅片上刻蚀出多孔阵列。
芯片入口不局限于一个入口，可以将入口设计为多个，每一个入口分别通入细胞溶液、培养溶液、药物溶液以及生物分子溶液等。
实施例3：
利用本发明基于微流控技术的多功能器官芯片可以构建人体器官，下面以人眼角膜为例进行说明：
一种基于微流控技术的多功能器官芯片来模拟人眼生理微环境的方法，包括以下步骤：
利用上述制作完成的器官芯片可以模拟人角膜微环境，此部分将详细介绍眼器官芯片的构建方法和操作步骤，主要包括芯片预处理、三维细胞培养、样品收集、分析表征，其流程图如图7所示。
子步骤C1，芯片预处理：
微流控芯片在超净台中用紫外照射灭菌大于等于30分钟，加入一型胶原蛋白修饰芯片，在37摄氏度条件下放置大于等于2小时，于超净台风干大于等于1小时，DMEM/F12培养基清洗大于等于3次，浸泡过夜，备用。
子步骤C2，三维细胞培养：
角膜内皮细胞消化后，将细胞悬液通过下层入口13将细胞接种至步骤C1中已修饰好的微流控芯片中，颠倒芯片，移入37摄氏度，5％二氧化碳培养箱中培养1天，角膜内皮细胞贴附于多孔膜8底面生长；再将角膜上皮细胞消化后，将细胞悬液通过第一细胞培养区5将细胞接种至微流控芯片中，移入37摄氏度，5％二氧化碳培养箱中培养1天，细胞贴附于多孔膜8界面生长为单层细胞，从第一细胞培养区5吸走液体，建立气液界面继续培养14天，使得角膜上皮细胞分化为多层细胞。期间利用注射泵进行灌流，下层入口13始终以一定速率持续灌注两 种细胞的混合培养基，同时下层出口14以相同速率稳定抽取，模拟人眼生理微环境。
子步骤C3，样品收集：
取出第一细胞培养区5的多孔膜8并通过下层出口14收集培养液。
步骤C4，分析表征：
对多孔膜8进行石蜡切片、冷冻切片，通过苏木精-伊红染色、免疫荧光染色等方法，利用倒置荧光显微镜、激光共聚焦显微镜观察三维培养的组织形态和生理学功能。对培养液进行检测，不仅可以通过聚合酶链式反应、实时荧光定量多聚核苷酸链式反应等方法检测生理和病理过程中的基因表达水平，还可以通过免疫细胞化学技术、蛋白免疫印迹、酶联免疫吸附等方法对蛋白表达进行定位、定性和定量研究，分析培养期间的关键因子、特征蛋白的变化。
器官芯片的细胞来源不局限于人眼来源的细胞，也可以是其他器官来源的原代细胞、癌细胞、多能干细胞、全能干细胞、单能干细胞等，能够在体外继续增殖分化即可。器官芯片构建的模型具有通用性，适用于不同人体器官，如脑、肺、心脏、肝脏、肾脏、肠道等。芯片预处理根据不同细胞来源，可选择不同型的胶原蛋白、纤维黏连蛋白、层黏连蛋白、基底膜蛋白或粘多糖等作为细胞外基质。
实施例4：一种基于微流控技术的多功能器官芯片来构建角膜损伤模型及其处理的方法，包括以下步骤：
步骤D，疾病模型：
利用上述构建完成的多功能眼器官芯片构建疾病模型，因此该步骤将详细介绍角膜损伤模型构建和操作步骤。模型建立主要包括疾病模型、药物处理、样品收集、分析表征，其流程图如图8所示。
子步骤D1，疾病模型：
利用200微升的移液器枪头，通过第一细胞培养区5划过眼器官芯片的多孔膜8，模拟由于环境因素或机械损伤造成的人角膜损伤。
子步骤D2，药物处理：
将间充质干细胞来源的细胞外囊泡作为促进角膜损伤修复的新型治疗药物，通过第一细胞培养区5加入，浸没角膜芯片，移入37摄氏度，5％二氧化碳中进行处理一段时间。
子步骤D3，样品收集：
取出第一细胞培养区5的多孔膜8并通过下层出口14收集培养液。
步骤D4，分析表征
对多孔膜8进行石蜡切片、冷冻切片，通过苏木精-伊红染色、免疫荧光染色等方法，利用倒置荧光显微镜、激光共聚焦显微镜观察三维培养的组织形态和生理学功能。对培养液进行检测，不仅可以通过聚合酶链式反应、实时荧光定量多聚核苷酸链式反应等方法检测生理和病理过程中的基因表达水平，还可以通过免疫细胞化学技术、蛋白免疫印迹、酶联免疫吸附等方法对蛋白表达进行定位、定性和定量研究，分析培养期间的关键因子、特征蛋白的变化。
步骤E，分析表征：
在步骤C2建立气液界面培养期间，可以通过第一细胞培养区5和第一测试区9测量跨上皮电阻值，评价角膜上皮屏障功能。获得上述构建完成的多功能眼器官芯片，根据步骤C4进行相关表征。获得上述构建完成的角膜损伤模型，根据步骤D4进行相关表征。
从图10中可以看到，角膜上皮细胞经芯片培养后形成多层细胞；图11中，角膜上皮细胞经芯片培养，随着培养时间的延长，跨上皮电阻值增加，侧面反映多层细胞且具有屏障功能；图12为角膜上皮细胞(A)和角膜内皮细胞(B)在芯片上培养，细胞形态正常且活细胞多。
根据基于微流控技术的多功能器官芯片，利用本发明模型构建方法还适用于构建涉及细胞迁移的生理过程，如胚胎发育、血管生成、伤口愈合、免疫反应、炎症反应、动脉粥样硬化、癌症转移等过程；药物可以是传统的小分子药物、大分子药物或新型的基因疗法、细胞疗法以及无细胞疗法等。
基于本发明基于微流控技术的多功能器官芯片，可以对人眼来源的细胞进行三维培养，形成近生理的三维结构，并考察了角膜屏障功能和关键蛋白表达。该模型可用于建立眼疾病模型、药物筛选、环境毒理学评价等，为探索人眼疾病的细胞形态学研究、分子机制及信号通路研究提供技术平台。
以上所述的具体实施例，对本发明的目的、技术方案和有益效果进行了进一步详细说明，应理解的是，以上所述仅为本发明的具体实施例而已，并不用于限制本发明，凡在本发明的精神和原则之内，所做的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (21)

  1. 一种基于微流控技术的多功能器官芯片，其特征在于：
    包括基底(3)，基底(3)上至少包括两个结构层，结构层上设有微通道以及与微通道连通的细胞培养区，相邻两个结构层的细胞培养区相连通，连通处设有多孔膜(8)。
  2. 根据权利要求1所述的一种基于微流控技术的多功能器官芯片，其特征在于：
    结构层上还设有测试区，所述测试区通过微通道与细胞培养区连通。
  3. 根据权利要求1所述的一种基于微流控技术的多功能器官芯片，其特征在于：
    结构层上的微通道(4)至少包括两个端口，一个为入口，一个为出口。
  4. 根据权利要求1所述的一种基于微流控技术的多功能器官芯片，其特征在于：
    所述多孔膜(8)为具有生物兼容性的多孔膜，且多孔膜(8)的孔径范围为0.1～10微米。
  5. 根据权利要求1-4任一所述的一种基于微流控技术的多功能器官芯片，其特征在于：
    结构层的材质为聚二甲基硅氧烷、玻璃、石英、硅片或聚甲基丙烯酸甲酯。
  6. 根据权利要求1-4任一所述的一种基于微流控技术的多功能器官芯片，其特征在于：
    结构层的厚度大于等于1毫米，微通道的深度大于等于200微米。
  7. 根据权利要求1-4任一所述的一种基于微流控技术的多功能器官芯片，其特征在于：
    细胞培养区的形状为圆形、矩形、三角形或者多边形。
  8. 根据权利要求1-4任一所述的一种基于微流控技术的多功能器官芯片，其特征在于：
    细胞培养区的数量为大于等于1个。
  9. 根据权利要求3所述的一种基于微流控技术的多功能器官芯片，其特征在于：
    结构层的数量为两层，分别为上层(1)、下层(2)；
    所述上层(1)内的微通道和细胞培养区分别为第一微通道(4)、第一细胞培养区(5)，下层(2)内的微通道和细胞培养区分别为第二微通道(6)、第二细胞培养区(7)，第一微通道(4)包括两个端口：上层入口(11)和上层出口(12)；第二微通道(6)包括两个端口：下层入口(13)和下层出口(14)；
    第一微通道(4)包括两个端口，一个为上层入口(11)，一个为上层出口(12)；第二微通道(6)至少包括两个端口，一个为下层入口(13)，一个为下层出口(14)。
  10. 一种基于微流控技术的多功能器官芯片的制备方法，其特征在于，包括以下步骤：
    1)制作各结构层，包括：制作结构层的微通道；再对细胞培养区和测试区进行钻孔，获得结构层；
    2)芯片键合，该步骤包含子步骤：
    2.1)将一个结构层与基底(3)键合；
    2.2)在已键合的结构层细胞培养区放置多孔膜(8)后，再将相邻两个结构层分别键合。
  11. 根据权利要求10所述的一种基于微流控技术的多功能器官芯片的制备方法，其特征在于：
    步骤1)中制作结构层的微通道采用PDMS翻模、干法刻蚀、湿法刻蚀或纳米压印方法制成。
  12. 根据权利要求10所述的一种基于微流控技术的多功能器官芯片的制备方法，其特征在于：
    步骤2)中，芯片键合的方式为氧等离子体键合、阳极键合或者熔融键合。
  13. 根据权利要求10所述的一种基于微流控技术的多功能器官芯片的制备方法，其特征在于：
    步骤1)制作各结构层具体包括：
    1.1)微通道阳模制作；
    1.2)材料浇筑；
    1.3)翻模，并制作微通道的入口和出口，再对细胞培养区和测试区进行钻孔，获得结构层。
  14. 根据权利要求13所述的一种基于微流控技术的多功能器官芯片的制备方法，其特征在于：
    步骤1.1)微通道阳模制作具体为：
    首先对硅片进行清洗并烘干，然后将硅片表面进行亲水化处理，并在表面旋涂一层光刻胶并前烘；光刻胶前烘后，利用微通道掩模版曝光并后烘；将后烘硅片基底浸入显影液中显影，并在一定温度下坚膜一段时间，使光刻胶硬化并增加其与硅片的粘附力，便可获得微通道阳模；
    步骤1.2)PDMS浇筑：
    将PDMS预聚物和PDMS固化剂按照一定质量比混合并搅拌均匀，然后将混合PDMS溶液浇筑到微通道阳模上，其中微通道阳模经过脱模剂预处理，并在一定温度下烘烤而使其固化；
    步骤1.3)PDMS翻模，翻模固化后的PDMS，并利用打孔器制作微通道的入口和出口；再次利用打孔器对第一细胞培养区(5)和测试区进行钻孔，便可获得PDMS结构层。
  15. 根据权利要求14所述的一种基于微流控技术的多功能器官芯片的制备方法，其特征在于：步骤2)具体为：
    步骤2.1)PDMS结构层与基底(3)键合：
    将PDMS结构层的底面和玻璃基底的顶面同时进行氧等离子体处理，然后将两者对准键合；
    步骤B8：相邻的PDMS结构层键合：
    将多孔膜(8)放置在下方的PDMS结构层的细胞培养区顶端；然后将下方PDMS结构层的底面和上方的PDMS结构层的顶面同时进行氧等离子体处理，最后将上下两PDMS结构层对准键合，依次完成两相邻PDMS结构层对准键合，便完成了微流控芯片的制作。
  16. 根据权利要求13所述的一种基于微流控技术的多功能器官芯片的制备方法，其特征在于：
    所述多孔膜(8)为聚碳酸酯膜或者硅片；当多孔膜(8)为硅片时，所述硅片上通过刻蚀方法刻蚀出多孔阵列。
  17. 一种基于微流控技术的多功能器官芯片来构建器官的方法，其特征在于，包括以下步骤：
    1)将权利要求1-9所述的基于微流控技术的多功能器官芯片预处理；
    2)三维细胞培养：
    提供器官组织中每一层细胞的细胞悬液，分别按次序通过微通道将细胞悬液注入各结构层中，放入培养箱中培养，使各层细胞贴附于多孔膜(8)生长，期间各层结构层可灌流各细胞的培养基。
  18. 一种基于微流控技术的多功能器官芯片来构建器官的方法，其特征在于，包括以下步骤：
    所述步骤1)中芯片预处理包括：对芯片进行灭菌，再加入细胞外基质修饰芯片，风干后用培养基清洗。
  19. 一种基于微流控技术的多功能器官芯片来构建人眼角膜的方法，其特征在于，包括以下步骤：
    步骤C1，将权利要求9所述的基于微流控技术的多功能器官芯片预处理；
    步骤C2，三维细胞培养：
    将角膜内皮细胞悬液通过下层入口(13)将细胞接种至芯片中，颠倒芯片，移入二氧化碳培养箱中培养，角膜内皮细胞贴附于多孔膜(8)底面生长；再将角膜上皮细胞悬液通过第一细胞培养区(5)将细胞接种至芯片中，移入二氧化碳培养箱中培养，细胞贴附于多孔膜(8)界面生长为单层细胞，从第一细胞培养区(5)吸走液体，建立气液界面继续培养，使得角膜上皮细胞分化为多层细胞，期间从下层入口(13)以一定速率持续灌注两种细胞的混合培养基，同时下层出口(14)以相同速率稳定抽取，模拟人眼生理微环境。
  20. 一种基于微流控技术的多功能器官芯片来构建眼器官芯片的方法，其特征在于，包括以下步骤：
    步骤C1，将权利要求9所述的基于微流控技术的多功能器官芯片预处理为：
    将芯片灭菌后，加入一型胶原蛋白修饰芯片，于超净台风干，DMEM/F12培养基清洗，浸泡过夜。
  21. 一种基于微流控技术的多功能器官芯片来构建角膜损伤模型及其处理的方法，其特征在于，包括以下步骤：
    步骤D1，疾病模型：
    基于权利要求19-20中构建的眼器官芯片，利用移液器枪头，通过第一细胞培养区(5)划过眼器官芯片的多孔膜(8)，模拟由于环境因素或机械损伤造成的人角膜损伤；
    步骤D2，药物处理：
    将促进角膜损伤修复的治疗药物，通过第一细胞培养区(5)加入，浸没角膜芯片，移入二氧化碳中进行处理；
    步骤D3，样品收集：
    取出第一细胞培养区(5)的多孔膜(8)并通过下层出口(14)收集培养液；
    步骤D4，分析表征
    对多孔膜(8)进行切片，观察三维培养的组织形态和生理学功能，对培养液进行检测，检测生理和病理过程中的基因表达水平；对蛋白表达进行定位、定性和定量研究，分析培养期间的关键因子、特征蛋白的变化。
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