CN112574884A - Multifunctional organ chip based on microfluidic technology, preparation method and application thereof - Google Patents
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Abstract
The invention belongs to the field of microfluidic technology and biotechnology, and relates to a multifunctional organ chip based on the microfluidic technology, a preparation method and application thereof. A multifunctional organ chip based on a microfluidic technology comprises a substrate, wherein the substrate at least comprises two structural layers, each structural layer is provided with a microchannel and a cell culture area communicated with the microchannel, the cell culture areas of two adjacent structural layers are communicated, and a porous membrane is arranged at the communication position. The invention provides a multifunctional organ chip based on a microfluidic technology, which not only can stably culture human corneal cells in a culture area to form a three-dimensional corneal tissue, but also can detect the integrity of an established corneal barrier, solve the problem that the human cornea is difficult to be correctly reflected by in vitro two-dimensional culture and in vivo animal experiments, and meet the requirements of scientific research and clinical application on in vitro bionic research models.
Description
Technical Field
The invention belongs to the field of microfluidic technology and biotechnology, and relates to a multifunctional organ chip based on the microfluidic technology, a preparation method and application thereof.
Background
The eye is the human visual organ, one of the most important organs in human sense, and is composed of the eyeball and its auxiliary structure, and about 80% of the information in the brain is obtained through the eye. With the improvement of economic level, the change of life style of people and the change of population structure in China, the disease spectrum of blinding eye diseases is changed greatly, and the main blinding eye diseases are changed from the past trachoma infectious eye diseases into metabolic and age-related non-infectious eye diseases mainly including cataract, keratopathy, retinopathy, ametropia, glaucoma and weak eyes, which are the key points of the challenge of eye health work in China and the future eye health work.
At the present stage, the research and development of pathophysiology of ophthalmic diseases and novel therapeutic drugs are mainly realized by two-dimensional cell culture models, animal models and human tissue donation. In 2019, the National Institutes of Health (NIH) proposed to accelerate the construction of three-dimensional ocular organs and in vitro micro-physiological systems, and in addition to more detailed summarization of the morphology and physiological response of human tissues in vivo, the method is expected to solve long-term scientific problems such as related tissue development, molecular signal conduction and pathophysiological mechanisms, and not only opens up a new way for research, but also provides a crucial guide for treatment development and patient care[1]。
In recent decades, organ chips based on microfluidic technology have been developed at a high speed, and gradually become an important platform for new generation of bionic research in the field of biomedicine due to the characteristics that the microenvironment is close to the physiological environment, accurate control can be provided in time and space dimensions, and multi-tissue function research is easy to realize. The use of microfluidic technology to study the effects of corneal epithelial barrier effects on the metabolism of eye drops has been reported[2]Effects of blinking on progression of dry eye[3]Effects of blinking shear force on corneal epithelial cell phenotype[4]. However, the device is not suitable for use in a kitchenHowever, none of the above models reduces the intact human corneal structure, which is crucial for the study of corneal microenvironment and drug metabolism in general ophthalmic diseases.
[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).
Disclosure of Invention
The invention provides a preparation method and application of a multifunctional organ chip based on a microfluidic technology, which can not only stably culture human corneal cells in a culture area to form three-dimensional corneal tissues, but also detect the integrity of an established corneal barrier, solve the problem that the human cornea is difficult to be correctly reflected by in-vitro two-dimensional culture and in-vivo animal experiments, and meet the requirements of scientific research and clinical application on in-vitro bionic research models.
The technical scheme for solving the problems is as follows: a multifunctional organ chip based on a microfluidic technology is characterized in that:
the device comprises a substrate, wherein the substrate at least comprises two structural layers, the structural layers are provided with microchannels and cell culture areas communicated with the microchannels, the cell culture areas of the two adjacent structural layers are communicated, and a porous membrane is arranged at the communication position.
Furthermore, a test area is arranged on the structural layer and is communicated with the cell culture area through a micro-channel.
Further, the micro-channel on the structural layer at least comprises two ports, one is an inlet and the other is an outlet.
Further, the porous membrane is a biocompatible porous membrane such as polycarbonate and polydimethylsiloxane, and the pore diameter of the porous membrane is in a range of 0.1 to 10 μm. The porous membrane can also be a silicon wafer, and a porous array is etched on the silicon wafer by an etching method.
Furthermore, the material of the structural layer is Polydimethylsiloxane (PDMS) glass, quartz, a silicon wafer, polymethyl methacrylate and other common micro-nano processing materials.
Furthermore, the thickness of the structural layer is greater than or equal to 1 millimeter, the thickness of the substrate is approximately equal to 500 micrometers, and the depth of the micro-channel is greater than or equal to 200 micrometers.
Further, the shape of the cell culture section is circular, rectangular, triangular or polygonal.
Further, the number of the cell culture sections is 1 or more.
Further, the number of the structural layers is two, namely an upper layer and a lower layer.
The microchannel and the cell culture area in the upper layer are respectively a first microchannel and a first cell culture area, the microchannel and the cell culture area in the lower layer are respectively a second microchannel and a second cell culture area, and the first microchannel comprises two ports: an upper inlet and an upper outlet; the second microchannel includes two ports: a lower inlet and a lower outlet.
The first microchannel comprises two ports, one is an upper inlet and one is an upper outlet; the second microchannel includes at least two ports, one being a lower inlet and one being a lower outlet.
In the invention, cells can be respectively inoculated to the upper surface and the lower surface of the porous membrane through the first microchannel of the upper layer and the second microchannel of the lower layer, and the cells cultured in the first cell culture area of the upper layer and the second cell culture area of the lower layer can realize material exchange through micropores on the porous membrane to form a corneal microenvironment to simulate human eye organs.
In addition, the invention also provides a preparation method of the multifunctional organ chip based on the microfluidic technology, which is characterized by comprising the following steps:
1) manufacturing each structural layer, including: manufacturing a micro-channel of the structural layer; drilling the cell culture area and the test area to obtain a structural layer;
2) chip bonding:
2.1) bonding a structural layer to the substrate;
2.2) after the porous membrane is placed in the cell culture area of the bonded structural layer, respectively bonding two adjacent structural layers.
Further, the micro-channel for fabricating the structural layer in step 1) is fabricated by using a PDMS mold-flipping, dry etching, wet etching or nanoimprint method.
Further, in the step 2), the die bonding mode is oxygen plasma bonding, anodic bonding or fusion bonding.
Further, the step 1) of manufacturing each structural layer specifically includes:
1.1) manufacturing a micro-channel male die;
1.2) pouring materials;
and 1.3) turning over the mold, manufacturing an inlet and an outlet of the micro-channel, and drilling the cell culture area and the test area to obtain a structural layer.
Further, the step 1.1) of manufacturing the micro-channel male die comprises the following steps:
firstly, cleaning and drying a silicon wafer, then carrying out hydrophilic treatment on the surface of the silicon wafer, and spin-coating a layer of photoresist on the surface and pre-drying; after the photoresist is pre-baked, exposing and post-baking by using a micro-channel mask; immersing the post-baked silicon wafer substrate into a developing solution for developing, and hardening the photoresist at a certain temperature for a period of time to increase the adhesion of the photoresist and the silicon wafer so as to obtain a micro-channel male die;
step 1.2) pouring PDMS:
mixing and uniformly stirring PDMS prepolymer and PDMS curing agent according to a certain mass ratio, and pouring the mixed PDMS solution onto a microchannel male mold, wherein the microchannel male mold is pretreated by a release agent and is baked at a certain temperature to be cured;
step 1.3), overturning the PDMS mold, namely overturning the cured PDMS, and manufacturing an inlet and an outlet of the microchannel by using a puncher; and drilling the first cell culture area and the test area by using the puncher again to obtain the PDMS structure layer.
Further, the step 2) is specifically:
step 2.1) bonding the PDMS structural layer with the substrate:
simultaneously carrying out oxygen plasma treatment on the bottom surface of the PDMS structure layer and the top surface of the glass substrate, and then aligning and bonding the two;
step B8: bonding of adjacent PDMS structural layers:
placing a porous membrane on the top of the cell culture zone of the underlying PDMS structural layer; and then, simultaneously carrying out oxygen plasma treatment on the bottom surface of the lower PDMS structure layer and the top surface of the upper PDMS structure layer, and finally aligning and bonding the upper PDMS structure layer and the lower PDMS structure layer to sequentially complete the aligning and bonding of the two adjacent PDMS structure layers, so that the micro-fluidic chip is manufactured.
Further, the porous membrane is a polycarbonate membrane or a silicon wafer; and when the porous membrane is a silicon wafer, etching a porous array on the silicon wafer by an etching method.
A method for constructing an organ by using a multifunctional organ chip based on a microfluidic technology is characterized by comprising the following steps:
1) chip pretreatment;
2) three-dimensional cell culture:
providing cell suspension of each layer of cells in organ tissues, injecting the cell suspension into each structural layer through the micro-channel in sequence, placing the structural layers into an incubator for culture, and enabling each layer of cells to be attached to the porous membrane for growth, wherein each structural layer can perfuse a culture medium of each cell.
Further, the step 1) chip pretreatment comprises: sterilizing the chip, adding extracellular matrix to modify the chip, air drying, and cleaning with culture medium.
In addition, the invention also provides a method for constructing the eye organ chip by using the multifunctional organ chip based on the microfluidic technology, which is characterized by comprising the following steps:
step C1, pre-treating the multifunctional organ chip based on microfluidic technology of claim 9;
step C2, three-dimensional cell culture:
after the corneal endothelial cells are digested, inoculating the cells into the chip through a lower layer inlet by using a cell suspension, inverting the chip, transferring the chip into a carbon dioxide incubator for culture, and attaching the corneal endothelial cells to the bottom surface of the porous membrane for growth; after the corneal epithelial cells are digested, inoculating the cells into a chip through a first cell culture area by using a cell suspension, transferring the chip into a carbon dioxide incubator for culture, attaching the cells to a porous membrane interface to grow into single-layer cells, sucking liquid from the first cell culture area, establishing a gas-liquid interface for continuous culture, differentiating the corneal epithelial cells into multi-layer cells, continuously perfusing a mixed culture medium of the two cells from a lower layer inlet at a certain speed all the time, and stably extracting from a lower layer outlet at the same speed to simulate a physiological microenvironment of human eyes.
Further, in the step C1, the multifunctional organ chip based on the microfluidic technology is preprocessed as follows: sterilizing the chip, adding collagen type I to modify the chip, air drying in a clean bench, washing with DMEM/F12 culture medium, and soaking overnight.
A method for constructing a corneal injury model and processing the corneal injury model by a multifunctional organ chip based on a microfluidic technology is characterized by comprising the following steps of:
step D1, disease model:
the human cornea microenvironment is simulated based on the construction of the steps C1 and C2,
utilizing a pipette tip to culture and divide the porous membrane passing through the ocular organ chip by the first cell, and simulating human corneal injury caused by environmental factors or mechanical injury;
step D2, drug treatment:
adding a therapeutic drug for promoting corneal injury repair through the first cell culture area, immersing the corneal chip, and transferring the corneal chip into carbon dioxide for treatment;
step D3, sample collection:
taking out the porous membrane of the first cell culture zone and collecting the culture solution through a lower outlet;
step D4, analysis and characterization
Slicing the porous membrane, observing the tissue morphology and physiological functions of three-dimensional culture, detecting culture solution, and detecting gene expression level in physiological and pathological processes; the protein expression is subjected to positioning, qualitative and quantitative research, and key factors and characteristic protein changes during culture are analyzed.
The invention has the advantages that:
1) compared with two-dimensional cell culture, the method can construct a three-dimensional cell culture model in vitro, and can more accurately simulate in-vivo microenvironment by accurately controlling parameters such as flow rate, concentration and components of the biological fluid;
2) the in-vitro bionic model constructed by human cells avoids the ethical problem and species difference problem of animal experiments all the time;
3) the invention constructs a complete human cornea structure, and can evaluate the integrity of the cornea barrier on a sheet;
4) a cornea injury disease model is constructed, and a novel research platform is provided for researching human eye disease development, drug metabolism and a novel treatment method;
5) the cell source of the organ chip is not limited to cells from human eyes, but can also be primary cells, cancer cells, pluripotent stem cells, totipotent stem cells, unipotent stem cells and the like from other organs, and the organ chip can be used for continuously proliferating and differentiating in vitro.
Drawings
FIG. 1 is a front view of a microfluidic chip designed according to the present invention;
FIG. 2 is a top view of a microfluidic chip designed according to the present invention;
FIG. 3 is a left side view of a microfluidic chip designed according to the present invention;
FIG. 4 is a three-dimensional view of a microfluidic chip designed according to the present invention;
FIG. 5 is a flow chart of the fabrication of a microfluidic chip;
FIG. 6 is a schematic diagram of a process for fabricating a microfluidic chip;
FIG. 7 is a flow chart of the construction of a multifunctional ocular organ chip;
FIG. 8 is a flow chart for constructing a disease model;
FIG. 9 is a diagram of a microfluidic chip in substance;
FIG. 10 is a graph showing the results of hematoxylin-eosin staining of corneal epithelial tissue in gas-liquid interfacial culture;
FIG. 11 shows the barrier function of corneal tissue cultured on an ocular organ chip;
FIG. 12 is a graph of calcein staining results of an ocular organ chip, wherein A is corneal epithelial tissue; and B is corneal endothelial tissue.
Wherein, 1, an upper layer, 2, a lower layer, 3, a substrate, 4, a first microchannel, 5, a first cell culture area, 6, a second microchannel, 7, a second cell culture area, 8, a porous membrane, 9, a first test area, 10, a second test area, 11, an upper layer inlet, 12, an upper layer outlet, 13, a lower layer inlet, 14 and a lower layer outlet.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be described clearly and completely with reference to the accompanying drawings of the embodiments of the present invention, and it is obvious that the described embodiments are some, but not all embodiments of the present invention. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention. Thus, the following detailed description of the embodiments of the present invention, presented in the figures, is not intended to limit the scope of the invention, as claimed, but is merely representative of selected embodiments of the invention.
A multifunctional organ chip based on a microfluidic technology is characterized in that:
the device comprises a substrate 3, wherein the substrate 3 at least comprises two structural layers, the structural layers are provided with microchannels and cell culture areas communicated with the microchannels, the cell culture areas of the two adjacent structural layers are communicated, and a porous membrane 8 is arranged at the communication position.
Preferably, the structural layer is further provided with a test area, and the test area is communicated with the cell culture area through a micro-channel.
In the present invention, the microchannel 4 on the structural layer comprises at least two ports, one is an inlet and the other is an outlet.
In the present invention, the porous membrane 8 is a porous membrane having biocompatibility such as a polycarbonate membrane or polydimethylsiloxane, and the pore diameter of the porous membrane 8 is in a range of 0.1 to 10 μm. The porous membrane can also be a silicon wafer, and a porous array is etched on the silicon wafer 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 and can be rectangles, triangles, polygons and the like, the sizes of the three-dimensional cell culture area and the test area are designed and adjusted according to the three-dimensional cell culture or tissue culture requirements, and the test area can be selectively reserved and deleted according to the test requirements; the number of the three-dimensional cell culture areas is not limited to one, different numbers can be set according to the flux requirement of the chip, and the three-dimensional cell culture areas can be separated from each other or can be connected in series. Preferably, in the present invention, the length, width, depth and other parameters of the microchannel are not limited to a specific size as long as the cell solution can be injected into the chip.
Example 1:
a multifunctional organ chip based on microfluidic technology, see fig. 1-4 and fig. 9. Referring to fig. 1, the chip comprises two structural layers, the upper layer 1 is Polydimethylsiloxane (PDMS) with a first microchannel 4, a first cell culture zone 5 and a first test zone 9, the lower porous membrane 8 is polycarbonate membrane with a microporous structure, the lower layer 2 is 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.
Referring to fig. 2, the circular holes at the upper left corner and the lower right corner of the chip of the invention are respectively the inlet and the outlet of a first microchannel 4, and the circular holes at the lower left corner and the upper right corner of the chip are respectively the inlet and the outlet of a second microchannel 6; the three-dimensional cell culture area in the middle of the chip is separated by a polycarbonate membrane to form a first cell culture area 5 and a second cell culture area 7 with the diameters of 2-10 mm; the test area in the middle of the chip is connected with the cell culture area through a micro-channel.
Referring to fig. 3, the thicknesses of the upper layer 1PDMS and the lower layer 2PDMS are greater than or equal to 1 mm, the thickness of the glass substrate is approximately equal to 500 micrometers, and the depths of the first microchannel 4 and the second microchannel 6 are greater than or equal to 200 micrometers; through the connection of the first microchannel 4 and the second microchannel 6, the test area is respectively communicated with the first cell culture area 5 and the second cell culture area 7, so that the physiological state of cells or tissues can be detected in real time in the cell culture process.
Referring to fig. 4, 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 zone 5, and a first test zone 9.
In this embodiment, the cells can be inoculated to 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, and the cells cultured in the first cell culture region 5 of the upper layer 1 and the second cell culture region 7 of the lower layer 2 can realize material exchange through the micropores of the porous membrane 8, so as to form a corneal microenvironment, which simulates human eye organs.
The invention relates to a multifunctional organ chip based on a microfluidic technology, which belongs to a microfluidic chip and has a unique structure, wherein the multifunctional organ chip comprises a three-dimensional cell culture area and a test area, can realize the co-culture of various cells or various tissues and can meet the requirement of detecting the growth states of the cells and the tissues in real time; meanwhile, the structure of the microfluidic chip has expandability, and the number, the shape and the layer number of the three-dimensional cell culture areas can be designed according to the experiment requirements.
Example 2:
a preparation method of a multifunctional organ chip based on a microfluidic technology comprises the following steps: taking two structural layers as an example:
the chip manufacturing process includes three parts, i.e., upper layer 1PDMS manufacturing, lower layer 2PDMS manufacturing, and chip bonding, as shown in fig. 5. The preparation method of the upper layer 1PDMS comprises the following steps: the method comprises the following steps of upper layer 1 microchannel male die manufacturing, upper layer 1PDMS pouring, upper layer 1PDMS mold overturning and upper layer 1PDMS punching, and is shown as a sub-graph S1 in figure 5. The preparation steps of the lower layer 2PDMS include: the fabrication of the micro-channel male mold of the lower layer 2, the pouring of the PDMS of the lower layer 2, the mold turnover of the PDMS of the lower layer 2 and the punching of the PDMS of the lower layer 2 are shown in the drawing S2 in FIG. 5. Then, bonding the lower layer 2PDMS with the glass substrate, sealing the second cell culture region 7 of the lower layer 2PDMS with the polycarbonate film, and finally aligning and bonding the upper layer 1PDMS with the lower layer 2PDMS to complete the manufacture of the microfluidic chip, as shown in fig. 5, sub-diagram S3.
Step S1: preparing an upper layer 1PDMS with a first microchannel 4, a first cell culture zone 5 and a first test zone 9, the steps comprising sub-steps B1-B3:
substep B1, male mold fabrication of the first microchannel 4:
firstly, cleaning and drying a silicon wafer, then carrying out hydrophilization treatment on the surface of the silicon wafer, spin-coating a layer of photoresist on the surface and then carrying out forward drying, wherein a sub-graph a in figure 6 is shown; after the photoresist is pre-baked, exposing and post-baking by using a micro-channel mask plate, as shown in a figure b in figure 6; and (3) immersing the post-baked silicon wafer substrate into a developing solution for developing, hardening the photoresist at a certain temperature for a period of time, and increasing the adhesive force between the photoresist and the silicon wafer to obtain a first microchannel 4 male die, which is shown as a sub-graph c in fig. 6.
Substep B2, casting upper layer 1 PDMS:
and mixing and uniformly stirring the PDMS prepolymer and the PDMS curing agent according to a certain mass ratio, and pouring the mixed PDMS solution onto the first micro-channel 4 male mold, wherein the first micro-channel 4 male mold is pretreated by a release agent and baked at a certain temperature to be cured, as shown in figure 6, panel d.
Substep B3, upper layer 1PDMS overmold:
turning over the cured PDMS, and making an inlet and an outlet of the first microchannel 4 by using a puncher, as shown in figure 6, panel e; the upper layer 1PDMS is obtained by drilling the first cell culture area 5 and the first test area 9 again with a 2-10 mm hole puncher, see panel f in FIG. 6.
Step S2: preparing a lower layer 2PDMS with a second microchannel 6, a second cell culture zone 7, and a second test zone 10, the steps including substeps B4-B6:
substep B4, male mold fabrication of the second microchannel 6:
firstly, cleaning and drying a silicon wafer, then carrying out hydrophilization treatment on the surface of the silicon wafer, spin-coating a layer of photoresist on the surface and then carrying out prebaking, wherein a graph g in figure 6 is shown; after the photoresist is pre-baked, exposing and post-baking by using a micro-channel mask plate, as shown in a graph h in fig. 6; and (3) immersing the post-baked silicon wafer substrate into a developing solution for developing, hardening the photoresist at a certain temperature for a period of time, and increasing the adhesive force between the photoresist and the silicon wafer to obtain a second microchannel 6 male die, which is shown as a figure i in figure 6.
Substep B5, lower layer 2PDMS casting:
and mixing and uniformly stirring the PDMS prepolymer and the PDMS curing agent according to a certain mass ratio, and pouring the mixed PDMS solution onto a second micro-channel 6 male mold, wherein the second micro-channel 6 male mold is pretreated by a release agent and is baked at a certain temperature to be cured, which is shown as figure j in figure 6.
Substep B6, lower layer 2PDMS overmold:
turning over the cured PDMS, and making an inlet and an outlet of the second microchannel 6 by using a puncher, as shown in figure 6, sub-figure k; the second cell culture and test areas were again drilled using a 2-10 mm hole punch to obtain the lower layer of 2PDMS, see panel l in FIG. 6.
Step S3, chip bonding, the step includes sub-steps B7-B8:
substep B7, bonding of the lower layer 2PDMS to the glass substrate:
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 both were aligned and bonded, see fig. 6, panel m.
Substep B8, bonding the upper layer 1PDMS with the lower layer 2 PDMS:
the polycarbonate membrane was placed in alignment on top of the second cell culture zone 7 in the lower layer 2PDMS, figure 6, panel n; and then, simultaneously carrying out oxygen plasma treatment on the bottom surface of the lower layer 2PDMS and the top surface of the upper layer 1PDMS, and finally aligning and bonding the two to complete the manufacture of the microfluidic chip, which is shown as a diagram o in fig. 6.
The manufacturing method of the multifunctional organ chip based on the microfluidic technology is simple and various, the manufacturing method of the micro-channel array comprises methods such as PDMS mold turning, dry etching, wet etching, nano imprinting and the like, and chip bonding can also be methods such as channel oxygen plasma bonding, anodic bonding, fusion bonding and the like.
The upper layer 1 and the lower layer 2 in the invention can be made of PDMS, silicon chip, quartz, glass and other materials by etching methods and other methods. The structural parameters of the micro-channel can be designed flexibly, such as depth, width, length and number, which can be designed or changed according to the requirements of cell culture and tissue culture.
When the chip manufacturing steps S1 and S2 are to manufacture the upper layer 1PDMS and the lower layer 2PDMS, the PDMS material may be replaced with a glass substrate or other materials such as polymethyl methacrylate, and then the microchannel array is etched at the bottom of the materials such as the glass substrate by using an etching method, or the microchannel array is manufactured at the bottom of the materials such as polymethyl methacrylate by using a nanoimprint method; in the chip manufacturing step S3, the chip bonding method is not limited to oxygen plasma bonding, and other bonding methods such as anodic bonding and fusion bonding may be selected according to the chip material; in the chip manufacturing step S3, the polycarbonate film may be replaced with a silicon wafer or other material, and then a porous array may be etched on the silicon wafer by an etching method.
The inlet of the chip is not limited to one inlet, and the inlet may be designed to be a plurality of inlets, and each inlet is filled with a cell solution, a culture solution, a drug solution, a biomolecule solution, and the like.
Example 3:
the multifunctional organ chip based on the micro-fluidic technology can be used for constructing human organs, and the cornea of the human eye is taken as an example for explanation:
a method for simulating a physiological microenvironment of a human eye by using a multifunctional organ chip based on a microfluidic technology comprises the following steps:
the organ chip manufactured by the method can simulate the human cornea microenvironment, and the construction method and the operation steps of the eye organ chip are described in detail in the section, and the method mainly comprises chip pretreatment, three-dimensional cell culture, sample collection, analysis and characterization, and the flow chart is shown in fig. 7.
Substep C1, chip pre-processing:
sterilizing the microfluidic chip in a super clean bench by ultraviolet irradiation for more than or equal to 30 minutes, adding a collagen modified chip, standing at 37 ℃ for more than or equal to 2 hours, air-drying in the super clean bench for more than or equal to 1 hour, cleaning with DMEM/F12 culture medium for more than or equal to 3 times, and soaking overnight for later use.
Substep C2, three-dimensional cell culture:
after the corneal endothelial cells are digested, inoculating the cells into the modified microfluidic chip in the step C1 through the lower layer inlet 13 by using the cell suspension, inverting the chip, transferring the chip into a 5% carbon dioxide incubator at 37 ℃ for culturing for 1 day, and attaching the corneal endothelial cells to the bottom surface of the porous membrane 8 for growth; and after the corneal epithelial cells are digested, inoculating the cells into the microfluidic chip by a cell suspension through the first cell culture area 5, transferring the microfluidic chip into a 37 ℃ carbon dioxide incubator for culture for 1 day, attaching the cells to the interface of the porous membrane 8 to grow into single-layer cells, sucking liquid from the first cell culture area 5, establishing a gas-liquid interface, and continuing to culture for 14 days to enable the corneal epithelial cells to be differentiated into multi-layer cells. During perfusion, the injection pump is used for perfusion, the lower layer inlet 13 continuously perfuses the mixed culture medium of two cells at a certain speed all the time, and the lower layer outlet 14 stably pumps at the same speed to simulate the physiological microenvironment of human eyes.
Substep C3, sample collection:
the porous membrane 8 of the first cell culture zone 5 is removed and the culture broth is collected through the lower outlet 14.
Step C4, analytical characterization:
the porous membrane 8 is subjected to paraffin sectioning and frozen sectioning, and the morphology and physiological function of the three-dimensionally cultured tissue are observed by an inverted fluorescence microscope, a laser confocal microscope, and the like by means of hematoxylin-eosin staining, immunofluorescence staining, and the like. The culture solution is detected, gene expression level in physiological and pathological processes can be detected by methods such as polymerase chain reaction, real-time fluorescence quantitative polynucleotide chain reaction and the like, protein expression can be localized, qualitatively and quantitatively researched by methods such as immunocytochemistry, western immunoblotting, enzyme-linked immunosorbent and the like, and changes of key factors and characteristic proteins during culture are analyzed.
The cell source of the organ chip is not limited to the cell from the human eye, but may be primary cells, cancer cells, pluripotent stem cells, totipotent stem cells, unipotent stem cells and the like from other organs, and the cell source can be obtained by continuous proliferation and differentiation in vitro. The model constructed by the organ chip has universality and is suitable for different human organs, such as brain, lung, heart, liver, kidney, intestinal tract and the like. According to different cell sources, the chip pretreatment can select different types of collagen, fibronectin, laminin, basement membrane protein or mucopolysaccharide and the like as extracellular matrixes.
Example 4: a method for constructing a corneal injury model and processing the corneal injury model based on a multifunctional organ chip of a microfluidic technology comprises the following steps:
step D, disease model:
the disease model is constructed by using the multifunctional eye organ chip constructed as described above, so the steps for constructing and operating the corneal injury model will be described in detail. The model establishment mainly comprises disease model, drug treatment, sample collection, analysis and characterization, and the flow chart is shown in figure 8.
Substep D1, disease model:
the human corneal injury due to environmental factors or mechanical damage was simulated by stroking the porous membrane 8 of the ocular organ chip through the first cell culture zone 5 using a 200 microliter pipette tip.
Substep D2, drug treatment:
the extracellular vesicles from the mesenchymal stem cells are used as a novel therapeutic drug for promoting corneal injury repair, are added through the first cell culture area 5, immerse the corneal chip, and are moved into 5% carbon dioxide at 37 ℃ for treatment for a period of time.
Substep D3, sample collection:
the porous membrane 8 of the first cell culture zone 5 is removed and the culture broth is collected through the lower outlet 14.
Step D4, analysis and characterization
The porous membrane 8 is subjected to paraffin sectioning and frozen sectioning, and the morphology and physiological function of the three-dimensionally cultured tissue are observed by an inverted fluorescence microscope, a laser confocal microscope, and the like by means of hematoxylin-eosin staining, immunofluorescence staining, and the like. The culture solution is detected, gene expression level in physiological and pathological processes can be detected by methods such as polymerase chain reaction, real-time fluorescence quantitative polynucleotide chain reaction and the like, protein expression can be localized, qualitatively and quantitatively researched by methods such as immunocytochemistry, western immunoblotting, enzyme-linked immunosorbent and the like, and changes of key factors and characteristic proteins during culture are analyzed.
And E, analyzing and characterizing:
during the establishment of the gas-liquid interface culture in step C2, the corneal epithelial barrier function can be evaluated by measuring the trans-epithelial electrical resistance value through the first cell culture zone 5 and the first test zone 9. The multifunctional eye organ chip constructed as described above was obtained and subjected to the relevant characterization according to step C4. And obtaining the constructed cornea damage model, and performing relevant characterization according to the step D4.
As can be seen from FIG. 10, corneal epithelial cells were cultured on a chip to form a plurality of layers of cells; in FIG. 11, corneal epithelial cells were cultured on a chip, and as the culture time was prolonged, the trans-epithelial electrical resistance value was increased, and the lateral surface reflected the multi-layered cells and had a barrier function; FIG. 12 shows that corneal epithelial cells (A) and corneal endothelial cells (B) were cultured on a chip, and the cells were normal in morphology and many viable cells were present.
According to the multifunctional organ chip based on the microfluidic technology, the model construction method is also suitable for constructing physiological processes related to cell migration, such as embryonic development, angiogenesis, wound healing, immune reaction, inflammatory reaction, atherosclerosis, cancer metastasis and other processes; the medicine can be traditional small molecule medicine, macromolecule medicine or novel gene therapy, cell-free therapy and the like.
Based on the multifunctional organ chip based on the microfluidic technology, cells from human eyes can be cultured in three dimensions to form a near-physiological three-dimensional structure, and the corneal barrier function and key protein expression are investigated. The model can be used for establishing eye disease models, drug screening, environmental toxicology evaluation and the like, and provides a technical platform for researching cell morphology research, molecular mechanism and signal path research of eye diseases.
The above-mentioned embodiments are intended to illustrate the objects, technical solutions and advantages of the present invention in further detail, and it should be understood that the above-mentioned embodiments are only exemplary embodiments of the present invention and are not intended to limit the present invention, and any modifications, equivalents, improvements and the like made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (21)
1. A multifunctional organ chip based on a microfluidic technology is characterized in that:
the device comprises a substrate (3), wherein the substrate (3) at least comprises two structural layers, the structural layers are provided with microchannels and cell culture areas communicated with the microchannels, the cell culture areas of the two adjacent structural layers are communicated, and a porous membrane (8) is arranged at the communication position.
2. The multifunctional organ chip based on the microfluidic technology according to claim 1, wherein:
the structural layer is also provided with a test area which is communicated with the cell culture area through a micro-channel.
3. The multifunctional organ chip based on the microfluidic technology according to claim 1, wherein:
the microchannel (4) on the structural layer comprises at least two ports, one is an inlet and the other is an outlet.
4. The multifunctional organ chip based on the microfluidic technology according to claim 1, wherein:
the porous membrane (8) is a biocompatible porous membrane, and the pore diameter range of the porous membrane (8) is 0.1-10 micrometers.
5. A multifunctional organ chip based on microfluidic technology according to any one of claims 1-4, characterized in that:
the material of the structural layer is polydimethylsiloxane, glass, quartz, silicon wafer or polymethyl methacrylate.
6. A multifunctional organ chip based on microfluidic technology according to any one of claims 1-4, characterized in that:
the thickness of the structural layer is more than or equal to 1 millimeter, and the depth of the micro-channel is more than or equal to 200 micrometers.
7. A multifunctional organ chip based on microfluidic technology according to any one of claims 1-4, characterized in that:
the cell culture zone is circular, rectangular, triangular or polygonal in shape.
8. A multifunctional organ chip based on microfluidic technology according to any one of claims 1-4, characterized in that:
the number of cell culture regions is 1 or more.
9. A multifunctional organ chip based on microfluidic technology according to claim 3, characterized in that:
the number of the structural layers is two, namely an upper layer (1) and a lower layer (2);
microchannel and cell culture district in upper strata (1) are first microchannel (4), first cell culture district (5) respectively, and microchannel and cell culture district in lower floor (2) are second microchannel (6), second cell culture district (7) respectively, and first microchannel (4) include two ports: an upper inlet (11) and an upper outlet (12); the second microchannel (6) comprises two ports: a lower inlet (13) and a lower outlet (14);
the first microchannel (4) comprises two ports, one being an upper inlet (11) and one being an upper outlet (12); the second microchannel (6) comprises at least two ports, one being a lower inlet (13) and one being a lower outlet (14).
10. A preparation method of a multifunctional organ chip based on a microfluidic technology is characterized by comprising the following steps:
1) manufacturing each structural layer, including: manufacturing a micro-channel of the structural layer; drilling the cell culture area and the test area to obtain a structural layer;
2) chip bonding, comprising the sub-steps of:
2.1) bonding a structural layer to the substrate (3);
2.2) after the porous membrane (8) is placed in the cell culture area of the bonded structural layer, respectively bonding two adjacent structural layers.
11. The method for preparing a multifunctional organ chip based on a microfluidic technology according to claim 10, wherein the method comprises the following steps:
the micro-channel for manufacturing the structural layer in the step 1) is manufactured by adopting a PDMS mold turning, dry etching, wet etching or nano-imprinting method.
12. The method for preparing a multifunctional organ chip based on a microfluidic technology according to claim 10, wherein the method comprises the following steps:
in the step 2), the chip bonding mode is oxygen plasma bonding, anodic bonding or fusion bonding.
13. The method for preparing a multifunctional organ chip based on a microfluidic technology according to claim 10, wherein the method comprises the following steps:
the step 1) of manufacturing each structural layer specifically comprises the following steps:
1.1) manufacturing a micro-channel male die;
1.2) pouring materials;
and 1.3) turning over the mold, manufacturing an inlet and an outlet of the micro-channel, and drilling the cell culture area and the test area to obtain a structural layer.
14. The method for preparing a multifunctional organ chip based on a microfluidic technology according to claim 13, wherein the method comprises the following steps:
step 1.1) the micro-channel male die is specifically manufactured as follows:
firstly, cleaning and drying a silicon wafer, then carrying out hydrophilic treatment on the surface of the silicon wafer, and spin-coating a layer of photoresist on the surface and pre-drying; after the photoresist is pre-baked, exposing and post-baking by using a micro-channel mask; immersing the post-baked silicon wafer substrate into a developing solution for developing, and hardening the photoresist at a certain temperature for a period of time to increase the adhesion of the photoresist and the silicon wafer so as to obtain a micro-channel male die;
step 1.2) pouring PDMS:
mixing and uniformly stirring PDMS prepolymer and PDMS curing agent according to a certain mass ratio, and pouring the mixed PDMS solution onto a microchannel male mold, wherein the microchannel male mold is pretreated by a release agent and is baked at a certain temperature to be cured;
step 1.3), overturning the PDMS mold, namely overturning the cured PDMS, and manufacturing an inlet and an outlet of the microchannel by using a puncher; and drilling the first cell culture area (5) and the test area by using the puncher again to obtain the PDMS structural layer.
15. The method for preparing a multifunctional organ chip based on a microfluidic technology according to claim 14, wherein the method comprises the following steps: the step 2) is specifically as follows:
step 2.1) bonding the PDMS structure layer with the substrate (3):
simultaneously carrying out oxygen plasma treatment on the bottom surface of the PDMS structure layer and the top surface of the glass substrate, and then aligning and bonding the two;
step B8: bonding of adjacent PDMS structural layers:
placing a porous membrane (8) on top of the cell culture zone of the underlying PDMS-structured layer; and then, simultaneously carrying out oxygen plasma treatment on the bottom surface of the lower PDMS structure layer and the top surface of the upper PDMS structure layer, and finally aligning and bonding the upper PDMS structure layer and the lower PDMS structure layer to sequentially complete the aligning and bonding of the two adjacent PDMS structure layers, so that the micro-fluidic chip is manufactured.
16. The method for preparing a multifunctional organ chip based on a microfluidic technology according to claim 13, wherein the method comprises the following steps:
the porous membrane (8) is a polycarbonate membrane or a silicon wafer; when the porous membrane (8) is a silicon wafer, a porous array is etched on the silicon wafer by an etching method.
17. A method for constructing an organ based on a multifunctional organ chip of a microfluidic technology is characterized by comprising the following steps:
1) pretreating the multifunctional organ chip based on the microfluidic technology of claims 1-9;
2) three-dimensional cell culture:
providing cell suspension of each layer of cells in organ tissues, respectively injecting the cell suspension into each structural layer through the micro-channel in sequence, putting the structural layers into an incubator for culture, and enabling each layer of cells to be attached to the porous membrane (8) for growth, wherein the structural layers can perfuse culture media of each cell.
18. A method for constructing an organ based on a multifunctional organ chip of a microfluidic technology is characterized by comprising the following steps:
the chip pretreatment in the step 1) comprises the following steps: sterilizing the chip, adding extracellular matrix to modify the chip, air drying, and cleaning with culture medium.
19. A method for constructing a cornea of a human eye by a multifunctional organ chip based on a microfluidic technology is characterized by comprising the following steps:
step C1, pre-treating the multifunctional organ chip based on microfluidic technology of claim 9;
step C2, three-dimensional cell culture:
inoculating the cells into the chip by the corneal endothelial cell suspension through a lower layer inlet (13), inverting the chip, transferring into a carbon dioxide incubator for culture, and attaching the corneal endothelial cells to the bottom surface of the porous membrane (8) for growth; inoculating the cells into the chip by the corneal epithelial cell suspension through the first cell culture area (5), transferring the chip into a carbon dioxide incubator for culture, attaching the cells to the interface of the porous membrane (8) to grow into single-layer cells, sucking liquid from the first cell culture area (5), establishing a gas-liquid interface for continuous culture, differentiating the corneal epithelial cells into multi-layer cells, continuously perfusing a mixed culture medium of the two cells from the lower layer inlet (13) at a certain speed, and stably extracting from the lower layer outlet (14) at the same speed to simulate the physiological microenvironment of human eyes.
20. A method for constructing an eye organ chip based on a multifunctional organ chip of a microfluidic technology is characterized by comprising the following steps:
step C1, pre-processing the microfluidic technology based multifunctional organ chip of claim 9 into:
sterilizing the chip, adding collagen type I to modify the chip, air drying in a clean bench, washing with DMEM/F12 culture medium, and soaking overnight.
21. A method for constructing a corneal injury model and processing the corneal injury model by using a multifunctional organ chip based on a microfluidic technology is characterized by comprising the following steps of:
step D1, disease model:
simulating human corneal damage due to environmental factors or mechanical damage by stroking the porous membrane (8) of the ocular organ chip through the first cell culture zone (5) using a pipette tip based on the ocular organ chip constructed in claims 19 to 20;
step D2, drug treatment:
adding a therapeutic drug for promoting corneal injury repair through the first cell culture area (5), immersing the corneal chip, and transferring the corneal chip into carbon dioxide for treatment;
step D3, sample collection:
removing the porous membrane (8) of the first cell culture zone (5) and collecting the culture solution through the lower outlet (14);
step D4, analysis and characterization
Slicing the porous membrane (8), observing the tissue morphology and physiological functions of three-dimensional culture, detecting culture solution, and detecting the gene expression level in the physiological and pathological processes; the protein expression is subjected to positioning, qualitative and quantitative research, and key factors and characteristic protein changes during culture are analyzed.
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