WO2022104626A1 - Puce d'organe multifonctionnelle basée sur la technologie microfluidique, son procédé de préparation et son utilisation - Google Patents
Puce d'organe multifonctionnelle basée sur la technologie microfluidique, son procédé de préparation et son utilisation Download PDFInfo
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- B—PERFORMING OPERATIONS; TRANSPORTING
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- 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
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- 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
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- 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
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- 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
Puce d'organe multifonctionnelle basée sur la technologie microfluidique, son procédé de préparation et son utilisation. La puce d'organe multifonctionnelle basée sur la technologie microfluidique comprend un substrat ayant au moins deux couches structurales disposées sur le substrat. Des microcanaux (4, 6) et des régions de culture cellulaire (5, 7) communiquant avec les microcanaux (4, 6) sont présents sur les couches structurelles, les régions de culture cellulaire (5, 7) de deux couches structurelles adjacentes sont en communication, et une membrane poreuse (8) est disposée au point de communication. La puce d'organe multifonctionnelle basée sur la technologie microfluidique permet non seulement de cultiver de manière stable des cellules de cornée humaine dans les régions de culture cellulaire (5, 7) et de former un tissu cornéen tridimensionnel, mais aussi de détecter l'intégrité d'une barrière cornéenne établie, de résoudre le problème de la difficulté de la culture bidimensionnelle in-vitro et des expériences animales in-vivo à reproduire correctement la cornée humaine, et de répondre aux exigences de la recherche scientifique et de l'application clinique sur le modèle de recherche bionique in-vitro.
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