CN117801951A - Multilayer barrier bionic chip and application and integrated system thereof - Google Patents
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Abstract
The invention provides a multilayer barrier bionic chip which sequentially comprises a micro-channel layer, a porous membrane layer, an upper chamber layer and a top cover layer from bottom to top, wherein the micro-channel layer and the upper chamber layer share a fluid space and provide a space for culturing lower cells, through holes are formed in the porous membrane layer and the upper chamber layer and are communicated with the micro-channel, the upper chamber layer is provided with a plurality of cell culture chambers, the bottom of each cell culture chamber is provided with a porous membrane of the porous membrane layer, the top cover layer covers the upper chamber layer and seals the through holes of the upper chamber layer, and cells on the inner side and the outer side of a simulated key functional interface are respectively cultured in the micro-channel layer and the upper chamber layer. The chip system of the invention can be applied to different scientific researches such as complex organ or multi-organ in vitro disease modeling, drug research and personalized medical treatment, and life science researches in special environments such as deep sea, space environment, high temperature and high pressure and strong light strong radiation environment, and provides feasible technical support.
Description
Technical Field
The invention relates to the field of chips for organ and/or cell culture, in particular to a multilayer barrier bionic chip and application and an integrated system thereof.
Background
Conventional life science research models are mainly based on animals and two-dimensional (2D) cells. While animal models have achieved human understanding of rich physiology and disease and development of new drugs, animal models have many limitations as models of human research, such as candidate drugs may be terminated by lack of efficacy on animals or by finding a hazard or toxicity on animals that may be unrelated to humans. Despite significant advances in vitro biology and toxicology over the last 20 years, currently more than 80% of study drugs fail in clinical trials, 60% of which are due to lack of efficacy and the other 30% are due to toxicity. 2D cell culture has been used for life science research for over a century. However, this culture approach is difficult to support tissue-specific differentiation functions of multiple cell types, and also difficult to provide information about the complexity of the life system. There is therefore an urgent need for in vitro modeling and testing platforms that can more truly reflect complex human features. In fact, with the rapid development of fine processing technology, bench-top experiments can be performed in small systems, promoting the development of microfluidic chip technology. The organ chip is a 3D microfluidic cell culture device, by means of the technology, and by combining a stem cell induced differentiation technology, a tissue engineering technology and the like, an organ physiological micro-system can be constructed, and the main structural functions of different tissues and organs of a human body and complex organ-to-organ connection can be simulated in vitro. The human organ chip technology is continuously favored by researchers, and can replace animal experiments in the future, thereby becoming a promising research means.
At present, most organ-chip studies are limited to in vitro modeling of individual organ functions (or functional interfaces), which ignores organ/tissue-organ barrier correlations, and in fact. Normal physiological operation of an organ requires physiological-related chemical processes such as digestion, absorption, operation, decomposition, metabolism, etc. of substances in the body, which are realized by body fluid and blood circulation. Furthermore, most of the analyses at this stage are based on off-chip and imaging techniques, which results in the operation of the organ-chip having to be coordinated with cumbersome application tools. The novel advanced multi-organ chip system has a remarkable trend in the development of drug research due to the evaluation of the modeling reduction degree of the drug effect, but the difficulty of operation analysis is further increased. With the progress of microfluidic technology, microfluidic driving and control of complex systems on a chip or based on the chip is provided with a device for replacing traditional large commercial equipment, and is more beneficial to the construction of an integrated system. Compared with the prior art, the biosensor provides sensitive, selective, on-site and real-time monitoring of microorganism physiological signals for diagnosis and treatment application related to tissue engineering, which greatly exploits the application scene of organ chips.
Disclosure of Invention
In order to overcome the problems in the prior art, the invention provides a multilayer barrier bionic chip, and an application and an integrated system thereof. The invention realizes in-vitro modeling of the target organ-organ barrier through structural design, and realizes integrated organ culture, biomedical detection, online fluid driving and control and in-situ observation platform by combining the in-vitro modeling design with a control chip module, a detection chip module and related matched equipment.
The invention provides the following technical scheme:
the multilayer barrier bionic chip comprises a micro-channel layer, a porous membrane layer, an upper chamber layer and a top cover layer from bottom to top in sequence, and is characterized in that the micro-channel layer and the upper chamber layer share a fluid space and provide a space for cell culture at the lower layer, through holes are formed in the porous membrane layer and the upper chamber layer and are communicated with the micro-channel, the porous membrane layer is provided with a porous membrane with the aperture of 8-15 mu m, the upper chamber layer is provided with a plurality of cell culture chambers, the bottom of each cell culture chamber is provided with a porous membrane of the porous membrane layer, the top cover layer seals the upper chamber layer and seals the through holes of the upper chamber layer, and the micro-channel layer and the upper chamber layer are used for respectively culturing cells at the inner side and the outer side of a simulated key functional interface.
Further, the micro-channel layer is made of glass, the height of the micro-channel is not less than 40 mu m, the length of the micro-channel corresponds to that of the upper cavity layer of the chip, the porous membrane layer is formed by clamping a porous membrane by a polydimethylsiloxane PDMS pore plate at the lower part and a glass pore plate at the upper part, the porous membrane is made of a polycarbonate membrane, and the upper cavity layer and the top cover layer are made of polymethyl methacrylate PMMA.
Further, a culture medium perfusion joint is arranged at a position corresponding to the culture chamber on the top cover layer, and liquid perfusion is carried out in the culture chamber through a liquid driving device.
Further, U87 human brain astrocytoma cells were cultured in the microchannel layer, hCMEC/D3 human brain microvascular endothelial cells were cultured in the first culture chamber in the upper chamber layer, SH-SY5Y neuroblastoma cells were cultured in the second culture chamber, and hCMEC/D3 human brain microvascular endothelial cells were cultured in the third culture chamber.
A method for constructing a neural-peripheral immune interaction model by using a multilayer barrier bionic chip, comprising the following steps:
step 1) sterilizing each structure of the chip, inoculating U87 cells to the micro-channel layer through the through hole of the upper chamber layer, filling the micro-channel layer and the upper chamber layer with a culture medium, covering the chip by using a top cover layer, clamping the top cover layer and the chip for inversion culture until the U87 cells are adhered to the porous membrane on the PDMS pore plate side of the porous membrane layer;
step 2) placing the front surface of the chip, discarding the culture medium, respectively inoculating hCMEC/D3 cells, SH-SY5Y cells and hCMEC/D3 cells to the first cell culture chamber, the second cell culture chamber and the third cell culture chamber of the upper chamber layer, carrying out cell perfusion culture on the first cell culture chamber and the third cell culture chamber after the cells are attached, wherein the perfusion rate is 500 mu L/h, and forming a compact structure by the hCMEC/D3 cells;
step 3) inoculating THP-1 human monocytic leukemia cells into the first cell culture chamber of the upper chamber layer.
Further: wherein the cell inoculation number ratio of U87 cells, SH-SY5Y cells, hCMEC/D3 cells and THP-1 cells is (3-10): 1: (5-20): (3-10).
An organ chip integrated system comprises an organ chip module, a liquid path control module and a detection chip module,
the organ chip module is characterized in that the multilayer barrier bionic chip in claim 3 is placed in a shell, and a temperature control fan and CO are communicated into the shell 2 The liquid inlet liquid paths of the perfusion connectors arranged in the first culture chamber and the third culture chamber of the upper chamber layer are connected with the injection pump, and the liquid outlet liquid paths are respectively connected with the first liquid storage device and the third liquid storage device;
the liquid path control chip of the liquid path control module is connected with the liquid outlet pipelines of the first liquid storage device and the third liquid storage device and the liquid outlet pipeline of the detection reserve liquid, the liquid inlet pipeline of the detection chip is connected with the liquid outlet pipeline of the detection chip, and the liquid path control chip is switched by the injection pump and the gas supply device;
the detection chip module is used for placing the detection chip on the objective table, the led-filtering base is arranged under the objective table, the small microscope is placed on the objective table, the light-shielding shell is arranged outside the objective table, the observation area is arranged on the light-shielding shell, the observation area corresponds to the ocular of the small microscope, the detection chip is connected in series by a plurality of reaction chambers, each reaction chamber is respectively connected with the liquid inlet pipeline, the functionalized carrier liquid inlet and the liquid outlet pipeline, and the liquid outlet pipeline is connected with the liquid storage device.
Further, the liquid path control chip is a basal layer, a PDMS buffer layer, a gas bin layer and a liquid path layer from bottom to top in sequence, the liquid path layer is a plurality of mutually communicated liquid branches, the gas bin layer is provided with a plurality of gas control units, each liquid branch corresponds to one gas control unit, the gas control units generate gas bin deformation to control the on-off of the liquid branch, the gas supply device is used for leading the gas in the gas storage bottle into the gas bin layer through a pressure control valve, and the electromagnetic valve is used for controlling the switching of the gas path.
Furthermore, the detection stock solution is respectively provided with buffer solution, a primary HCR amplification probe and a secondary HCR initiation probe stock solution, a secondary HCR amplification fluorescent probe stock solution, and the detection stock solution is connected into the liquid path control chip by a four-way pipe.
Further, the liquid inlet pipeline of the detection chip is filled with the culture solution flowing out of the first liquid storage device and the third liquid storage device and the detection reserve liquid, a plurality of functional hydrogel particles are embedded in the reaction chamber, and the aperture of the liquid inlet pipeline of the detection chip is smaller than the particle size of the functional hydrogel particles.
By adopting the technical scheme, the invention has the following beneficial effects:
(1) The barrier bionic chip can realize in-vitro modeling of multicellular co-culture, simulate complex and dynamic microenvironment required by cells, realize static or dynamic multicellular multi-type cell co-culture, provide effective research on interaction among cells, and can be repeatedly used and used as a diversified universal platform.
(2) The barrier bionic chip simplifies operation, reduces pollution risk and reduces requirements on operators and complex instruments. The experiment can be completed quickly, and the cells are returned to a stable and proper growth environment.
(3) The barrier bionic chip can provide a compact processing method of various materials (PMMA, PDMS, glass), and provides a specific technical method for chip design application aiming at different material characteristics.
(4) The barrier bionic chip system of the invention not only can be applied to different scientific researches such as complex organ or multi-organ in vitro disease modeling, drug research and development, personalized medical treatment and the like, but also can be applied to life science researches in special environments such as deep sea, space environments, high temperature and high pressure, strong light and strong radiation environments, and provides feasible technical support.
(5) The detection method in the barrier bionic chip system of the invention innovatively combines the functionalized hydrogel as a solid phase carrier and the micro-fluidic chip technology, and can provide a plurality of probe binding points and methods for identifying/capturing biomarkers on the chip, thereby providing more possibility for the timely detection technology.
(6) According to the two-stage HCR method design of the detection method in the barrier bionic chip system, the second-stage HCR probe is designed to be universal, the two-stage HCR can be amplified only by designing the first-stage HCR probe according to the target, and the target concentration is converted into a fluorescent signal through a fluorescent reporter molecule of the second-stage HCR.
Drawings
FIG. 1 is a schematic diagram of the structure of a multi-layered cultured organ barrier chip according to the invention;
FIG. 2 is a diagram showing an inverted culture of a material without bubbles in example 2 of the present invention;
FIG. 3 is a graph showing the results of an adherent cell bright field imaging experiment in example 2 of the present invention;
FIG. 4 is a graph showing the results of the resistivity test between the first culture chamber and the second culture chamber in example 2 of the present invention;
FIG. 5 is a graph showing the results of permeation rates of FITC-dextran tracers (4 kDa,20kDa,70 kDa) of different molecular weight sizes in a neurovascular unit barrier model constructed on a barrier chip in example 2 of the present invention;
FIG. 6 is an immunofluorescence staining chart of a tight junction protein of static culture and perfusion culture in a neurovascular unit barrier model constructed on a barrier chip in example 2 of the present invention;
FIG. 7 is a schematic diagram showing the structure of an organ-chip integration system according to embodiment 3 of the invention;
FIG. 8 is a schematic diagram showing the connection of the liquid path of the organ-chip integrated system according to embodiment 3 of the invention;
FIG. 9 is a schematic diagram showing the structure of a control chip in embodiment 4 of the present invention;
FIG. 10 is a schematic diagram showing the connection of air paths of a control chip module in embodiment 4 of the present invention;
FIG. 11 is a schematic diagram of a deformation structure of a control chip module in embodiment 4 of the present invention;
FIG. 12 is a schematic view of interface connection of a flow channel layer of a control chip module in embodiment 4 of the present invention;
FIG. 13 is a schematic diagram showing the structure of a detection chip module in embodiment 5 of the present invention;
FIG. 14 is a schematic diagram showing the structure of a detection chip in embodiment 5 of the present invention;
FIG. 15 is a schematic diagram of HCR reaction principle of the detection chip in example 5 of the present invention;
FIG. 16 is a graph showing the experimental results of miRNA-21 and miRNA-let7a detection performed by the detection chip in example 5 of the present invention on the medium in the first culture chamber (blood inlet) and the third culture chamber (blood outlet) of the barrier chip.
Description of the reference numerals
101. Joint, 102, top cover layer, 103, upper chamber layer, 104, glass well plate, 105, porous membrane, 106, PDMS well plate, 107, microchannel layer, 108, top cover layer for stationary culture, 109, top cover layer for perfusion culture, 1010, through-hole, 201, channel layer, 202, gas cartridge layer, 203, PDMS buffer layer, 204, base layer, 205, gas control unit, 206, liquid branch, 207, gas control unit, 208, control valve, 209, gas cylinder, 2010, solenoid valve, 301, stage, 302, filter, 303, microscope, 304, light-shielding housing, 305, observation area, 306, led light source, 307, filter, 407, liquid inlet line, 408, 409, functionalized carrier liquid inlet, 4010, liquid outlet line.
Detailed Description
The present invention will be described in further detail with reference to the drawings and examples, in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the drawings and detailed description are only intended to illustrate the invention and are not intended to limit the invention.
Example 1
As shown in fig. 1, a multilayer barrier bionic chip sequentially comprises a micro-channel layer 107, a porous membrane layer, an upper chamber layer 103 and a top cover layer 102 from bottom to top, wherein through holes 1010 are formed in the porous membrane layer and the upper chamber layer and are communicated with micro-channels, and the micro-channel layer and the upper chamber share a fluid space and provide a space for culturing lower cells.
The porous membrane layer is provided with a porous membrane 104 with the aperture of 8-15 mu m, the upper chamber layer is provided with a plurality of cell culture chambers, the culture of upper cells is carried out in the cell culture chambers, the bottom of the cell culture chambers is the porous membrane of the porous membrane layer, the top cover layer seals the upper chamber layer and seals the through holes of the upper chamber layer, and the cells on the inner side and the outer side of the simulated key functional interface are respectively cultured in the micro-channel layer and the upper chamber layer. The multi-layer barrier bionic chip is formed by a porous membrane which can pass through biological information of cell-cell interaction, prevent cell seeds from passing through and support cells from growing on the wall.
The micro-channel layer is made of glass, the height of the micro-channel is not less than 40 mu m, the length of the micro-channel corresponds to that of the upper cavity layer of the chip, the upper cavity of the chip and the left and right ends/inlets of the upper cavity are covered, the porous membrane layer is formed by clamping a porous membrane 104 by a Polydimethylsiloxane (PDMS) pore plate 105 at the lower part and a glass pore plate 103 at the upper part, the porous membrane is made of a polycarbonate membrane, and the upper cavity layer and the top cover layer are made of polymethyl methacrylate (PMMA).
The top cover layer is detachable, and can adopt a top cover layer 108 for static culture or a top cover layer 109 for perfusion culture, and a culture medium perfusion connector 101 is arranged at a position corresponding to a culture chamber of the top cover layer during perfusion culture, and liquid perfusion is carried out in the culture chamber through a liquid driving device.
U87 human brain astrocytoma cells are cultured in a microchannel layer, hCMEC/D3 human brain microvascular endothelial cells are cultured in a first culture chamber in an upper chamber layer, SH-SY5Y neuroblastoma cells are cultured in a second culture chamber, and hCMEC/D3 human brain microvascular endothelial cells are cultured in a third culture chamber, capable of mimicking neuro-peripheral immune interactions.
In the processing of the multi-layered cultured organ barrier chip of the present invention, the 4-layered structure is processed in advance, respectively, and then bonding (PDMS and glass) or sticking (glass and PMMA) is performed on two layers by two layers.
Firstly, the specific operation steps of the etching process of the glass micro-channel are as follows: (1) cleaning the mask: ensuring that the mask is not broken or scratched. Special attention should be paid to the integrity of the channels. It is necessary that the glass chrome plate be prepared in advance so that channels can be etched in the glass. (2) photolithography: the mask was placed on the adhesive side of the chromium plate under light-shielding conditions and subjected to photolithography for 30 seconds (as the case may be). (3) developing: developed with 0.8% naoh solution and gently shaken for 2 min. (4) fixing: baking in an oven at 110 ℃ for 15 minutes. (5) dechroming: soaking the chromium removing liquid for 1min, opening a bow left and right during chromium removing, and slightly shaking the chromium removing liquid back and forth (6) for chemical etching: before etching, yellow adhesive tape is used to wind the chromium plate, the etching liquid should not exceed the chromium plate, and the etching liquid is vibrated in water bath at 40 ℃ until the vibration direction is consistent with the direction of the etching channel, and the etching is performed for about 30 min. (7) secondary dechroming: taking down the yellow adhesive tape wound on the chromium plate, taking the adhesive tape, so as to avoid breaking the chromium plate by excessive force or carelessly scratching the chromium plate, taking down the adhesive tape, and removing the glue on the surface by using acetone. The chromium plate was placed in the dechroming solution and gently shaken until the chromium layer was removed. Punching the grid plate: firstly, cleaning and drying a chromium plate, and adhering the chromium plate to a thick glass plate by using 502 glue. (8) glue boiling: boiling water in an electromagnetic oven to cook thick glass and chromium plate after punching, boiling water at 120 deg.c, adding water once for 30min until the chromium plate and the glass plate are separated. (9) activation: the etched glass microchannel layer was activated overnight in concentrated sulfuric acid. (10) desulfuric acid: for the subsequent cell culture, the glass microchannel layer activated in concentrated sulfuric acid was washed with clean water for 30s, then immersed in absolute ethanol for 2h, and then washed with ultrapure water for 30s. Drying with nitrogen and baking for 1h at 60 ℃. (11) standby: the surface is covered by the adhesive tape and can be stored for standby.
Secondly, the sandwich porous membrane is processed mainly by punching the upper glass and the lower PDMS respectively, then plasma treating the lower PDMS for 27s, placing the cut porous membrane in alignment with the punching position, and then pressing and bonding the bonding surface of the upper glass and the lower PDMS with the porous membrane by plasma treating for 27 s.
In addition, the PMMA top cover layer and the upper cavity layer are custom-made by machining, and are subjected to ultrasonic cleaning (60 min) by filling ultrapure water before bonding or adhesion of the layers, soaking in absolute alcohol for 6h, flushing with ultrapure water (30 s) and drying with nitrogen. And then the upper cavity is packaged by transparent adhesive tape for standby. The top layer was then poured with PDMS and left to cure in an oven at 50℃for 4 hours.
The sandwich porous membrane is then bonded to the glass microchannel layer. The PMMA upper chamber and upper glass layer of the sandwich porous membrane were then glued together with 3M double sided tape, clamped with a clamp, and left overnight at room temperature.
PDMS pre-solution was prepared prior to use: curing agent=15:1, the mixture was poured into the closed cap layer, the above chamber was clamped between the mold and the cap layer for perfusion, the mixed PDMS was perfused, and the mixture was placed in an oven, and molded at 50 ℃ for 4 hours. This pretreatment can prevent the occurrence of a liquid leakage phenomenon at the time of inversion culture or at the time of perfusion.
Example 2
The invention provides a method for constructing a nerve-peripheral immune interaction model by utilizing organ chips cultured in multiple layers, which comprises the following steps:
step 1) sterilizing each structure of the chip, inoculating U87 cells to the micro-channel layer through the through hole of the upper chamber layer, filling the micro-channel layer and the upper chamber layer with a culture medium, covering the chip by using a top cover layer, clamping the top cover layer and the chip for inversion culture until the U87 cells are adhered to the porous membrane on the PDMS pore plate side of the porous membrane layer;
step 2) placing the front surface of the chip, discarding the culture medium, respectively inoculating hCMEC/D3 cells, SH-SY5Y cells and hCMEC/D3 cells to the first cell culture chamber, the second cell culture chamber and the third cell culture chamber of the upper chamber layer, carrying out cell perfusion culture on the first cell culture chamber and the third cell culture chamber after the cells are attached, wherein the perfusion rate is 500 mu L/h, and forming a compact structure by the hCMEC/D3 endothelial cells;
step 3) inoculating THP-1 human monocytic leukemia cells into the first cell culture chamber of the upper chamber layer.
Wherein the cell inoculation number ratio of U87 cells, SH-SY5Y cells, hCMEC/D3 cells and THP-1 cells is (3-10): 1: (5-20): (3-10).
First, a critical barrier function interface of neurovascular units is built on chip. After 12h overnight inversion culture, the capping layer-based PDMS sealing layer served as a buffer layer in contact with the chip, which can prevent leakage and also can prevent the growth of cells from being affected by the generation of large bubbles during inversion due to the hydrophobicity of PDMS and slow operation (fig. 2). As shown in fig. 3, the adherent condition of adherent cells can be observed by microscopic imaging. The results showed that both U87 and HCMEC/D3 cultured on both sides of the membrane in the first culture chamber grew, exhibited a healthy growth state, and both U87 cells exhibited a slender shape at both ends of the cell body, and HCMEC/D3 cells exhibited a typical epithelial-like shape. Cells can also be seen to grow in the second culture chamber in a healthy state of growth, with the U87 cells growing as described above, and SH-SY5Y cells being epithelially and growing in a clump. Also the cell growth of the third culture chamber is identical to that of the first unit. Therefore, the feasibility of the inoculation mode on the chip is verified.
The resistivity (the resistivity of the medium was subtracted) between the first culture chamber and the second culture chamber within 40h after all cells were inoculated by using a 24-well plate-sized transmembrane resistor, and as a result, as shown in fig. 4, the resistivity of the perfusion culture from 30h was significantly higher than the static culture resistivity, 162.3%,30.6%, and 90.1%, respectively, and the resistivity of 30h was already greater than 1000 Ω, indicating that the barrier integrity was good, and the values for drug metabolism testing were possessed.
FITC-dextran tracers of various sizes are commonly used to assess membrane barrier permeability in vivo and in vitro. Three common sizes of tracer-sized permeabilities were selected for this experiment to study the barrier permeability of neuro-vascular units, tracer was added to the upstream blood inlet simulation chamber (upper chamber of the first culture chamber) and allowed to stand for 24h. The results are shown in FIG. 5, although the porous polycarbonate membrane and the matrix coating itself have high permeability to 4kDa-70kDa FITC-dextran, permeability is 10 -4 cm 2 S, but has very low permeability to these large tracer molecules on chip that constructs the blood brain barrier. Wherein the permeability (P Permeability of ) At apparent permeability coefficient of 10 -5 cm 2 /s, but both 20kDa and 70kDa reach 10 -7 -10 -8 On the order of cm/s, in addition, the permeability exhibits a result inversely proportional to the stokes radius of FITC-dextran. When the corresponding stokes radius increases from 1.4 nanometers (4 kDa) to 6 nanometers (70 kDa), the permeability of FITC-dextran decreases by 95.2%. Has such strong affinity for hydrophilic nonionic tracersIndicating that tight junctions complexes are properly formed between adjacent endothelial cells. The overall low permeability and size dependence on FITC-dextran further demonstrates the barrier integrity of the neurovascular units constructed in our model.
One prominent feature of the barrier function of neurovascular units is the unique tightness between endothelial cells, which limits paracellular transport across the barrier of neurovascular units. The compactness of this barrier is supported by a well-designed network of intercellular connections (mainly tight junctions). Analysis of the barrier tightness of the barrier model of neurovascular units we examined the expression of the Tight Junction (TJ) protein (claudin-5, zo-1). Immunostaining showed high levels of protein expression in both claudin-5 and ZO-1 in static as well as perfusion cultures (fig. 6). In contrast, however, hCMEC/D3 cells undergoing dynamic perfusion culture not only exhibit more pronounced protein expression, but also the cells have a tendency to cascade growth, which may be related to perfusion conditions that can provide complex spatial nutrient exchanges, thereby enhancing metabolic activity of the cells, including transport of oxygen and nutrients, and waste removal. In addition, cells exhibit cell morphology that grows in the same direction, since the shear stress of perfusion provides the cells with a dominant direction, and is also one of the important reasons for improving barrier integrity.
Example 3
As shown in fig. 7 and 8, the invention further provides an organ chip integration system, which comprises an organ chip module, a liquid path control module and a detection chip module, wherein the integration system is used for providing cell culture matched with the organ chip, detection of biomarkers and liquid control of the system, and all the modules are connected into the integration system through reserved clamping grooves, positioning glue or clamping positions.
As shown in FIG. 8, the organ-chip module places the organ chips cultured in multiple layers in a housing, and communicates a temperature-controlled fan and CO into the housing 2 The liquid inlet paths of the perfusion joints d and e arranged in the first culture chamber and the third culture chamber of the upper chamber layer 1 are connected with the injection pump 13, and the liquid outlet paths d and e are respectively connected with the first liquid storage device 6 and the third liquid storage device7。
The shell adopts a light-transmitting PMMA square shell, and a temperature control fan, a shockproof sponge cushion and a multilayer cultured organ chip which are connected by a motor are assembled in the shell. Punching under the shell and connecting a conveyor of 5% CO 2 Is a gas cylinder of (2). And a chip with a temperature control belt display is connected through a wall-crossing connector, and a motor and a plug wire are connected for connecting a power supply and controlling the temperature (37 ℃) in the bin.
To facilitate independent operation and viewing of the organ-chip, the organ-chip module may be made detachable, with a transparent housing adapted to the stage of an inverted microscope, allowing direct viewing under the microscope. In addition, the upstream and downstream of the connected organ chip are connected by the quick connector CPC with the valve and the filter membrane, and besides the whole organ chip module can be placed under a microscope for long-term observation, the organ chip can be independently disassembled for operation and observation.
The liquid path control chip of the liquid path control module is connected with the liquid outlet pipelines n and 0 of the first liquid storage device and the third liquid storage device at the upstream and the liquid outlet pipeline p for detecting the stock solution, and is connected with the liquid inlet pipe u of the detection chip at the downstream, and the liquid path control chip completes the switching of the liquid paths by the injection pump 3 and the gas supply device. The liquid path control module transmits the culture medium and the detection stock solution in the first liquid storage device and the third liquid storage device to the detection chip module.
The detection chip module is used for placing the detection chip on the objective table, the led-light filtering base is arranged under the objective table, the small microscope is placed on the objective table, the light-shielding shell is arranged outside the objective table, the observation area is arranged on the light-shielding shell and corresponds to the ocular of the small microscope, the detection chip is connected in series by a plurality of reaction chambers, each reaction chamber is respectively connected with the liquid inlet pipeline, the functionalized carrier liquid inlet and the liquid outlet pipeline, and the liquid outlet pipeline is connected with the liquid storage device. A plurality of required biological tests are carried out in a test chip module, and in-situ observation on the test chip is carried out.
Example 4
The liquid path control module is connected with the liquid path control chip and the related liquid path and gas path pipelines. As shown in fig. 9-12, the liquid path control chip sequentially comprises a substrate layer 204, a PDMS buffer layer 203, a gas bin layer 202 and a liquid path layer 201 from bottom to top, the liquid path layer is a plurality of mutually communicated liquid branches, the gas bin layer is provided with a plurality of gas control units 205, each liquid branch 206 corresponds to one gas control unit 207, gas bin deformation occurs to control on-off of the liquid branch, the gas supply device leads the gas in the gas storage bottle 209 into the gas bin layer through a pressure control valve 208, and the electromagnetic valve 2010 controls switching of a gas path. Four solenoid valves and a switch and a connecting pipeline connected with the control chip. Four solenoid valves can control two chips respectively, 8 groups of channels (two groups of units on a control chip) can be added if more channels need to be controlled simultaneously.
As shown in fig. 9 and 12, the chip is three separate control units, each of which includes four independent gas reservoirs for introducing gas to create membrane type changes to intercept the fluid in the upper cross fluid path. The liquid channel comprises a liquid channel layer, a liquid outlet pipeline, a liquid inlet pipeline, a liquid outlet pipeline, a buffer liquid storage liquid, a liquid pump, a liquid inlet pipeline, a liquid channel layer and a liquid channel layer, wherein the No. 1 hole site is correspondingly connected with the liquid outlet pipeline of the first liquid storage device, the No. 2 hole site is correspondingly connected with the liquid outlet pipeline of the third liquid storage device, the No. 4 hole site is connected with the liquid outlet pipeline of the detection storage liquid, the No. 6 hole site is connected with the buffer liquid storage liquid, the No. 3 hole site is connected with the liquid inlet pipeline of the detection chip, and the No. 5 hole site is connected with the injection pump. According to the detection flow, the passage of the No. 3 hole site is blocked, and the upstream liquid to be detected is sucked into the injection pump connected with the No. 5 hole site from the No. 1/No. 2 hole site. And then opening the hole No. 3, blocking the hole No. 1/2 passage, and continuously filling the to-be-detected liquid for 1h into the detection chip connected with the hole No. 3 through the injection pump connected with the hole No. 5. Then the passages of the holes 3 and 6 are blocked, and the detection stock solution connected with the hole 4 is sucked into the injection pump connected with the hole 5. Then opening the hole No. 3 and blocking the hole No. 4, and injecting the detection liquid into the detection chip connected with the hole No. 3 at the flow rate of 2 mu L/min for 1h. Then the passage of the No. 4 hole site is blocked, the No. 6 hole site is opened, and the buffer stock solution connected with the No. 6 hole site is sucked into the injection pump connected with the No. 5 hole site. And then turning off the hole No. 6, and pouring the buffer solution to a detection chip connected with the hole No. 3 by a syringe pump connected with the hole No. 5, wherein the flow rate is set to be 2 mu L/min, and the pouring time is set to be 5min.
Example 5
As shown in fig. 13, the detection chip module places the detection chip on a stage 301, a led-filtering base including a 488nm optical filter 302 and a led light source 306 is arranged under the stage, a small microscope 303 is placed on the stage, a light-shielding shell 304 is arranged outside the stage, an observation area 305 is arranged on the light-shielding shell, and an 525nm optical filter 307 is arranged at the observation area for filtering stray light, and an eyepiece of the small microscope. The detection chip module is arranged at the most downstream of the integrated chip platform, and can be matched with the detection chip and read the concentration information of miRNA by combining a fluorescence imaging system to convert the information into a fluorescence image.
The large space and the visual window of the detection chip module can generate interference when observing fluorescent signals on the chip, so that an independent detection chip observation platform is constructed. The platform consists of a chip base with a light source clamping groove, a green LED light source, a light-resistant resin shell capable of holding a mobile phone for capturing pictures, a 40X amplification microscope and a detachable power line. When the fluorescent light source is used, firstly, the chip is connected with the access, then the LED light source is connected with the power supply, then the shell is opened, the chip is placed on the base, the hole to be tested is aligned with the light source, then the magnifying microscope is aligned with the hole to be tested, then the shell is found, the mobile phone and the capturing hole are aligned with the ocular lens of the microscope, and after the reaction is completed, the light source switch is turned on to capture fluorescent pictures. Fluorescence intensity analysis was then performed with image J.
As shown in fig. 14, the detection chip is connected in series by a plurality of reaction chambers 408, each of which is respectively connected to a liquid inlet pipe 407, a functionalized carrier liquid inlet 409 and a liquid outlet pipe 4010, and the liquid outlet pipe is connected to a liquid storage device. The liquid inlet pipeline of the detection chip is filled with culture solution flowing out of the first liquid storage device and the third liquid storage device and detection reserve liquid, a plurality of functional hydrogel particles are embedded in the reaction chamber, and the aperture of the liquid inlet pipeline of the detection chip is smaller than the particle size of the functional hydrogel particles.
Each reaction chamber corresponds to 1 liquid inlet pipeline 407,1 functionalized carrier liquid inlet 409 and 1 liquid outlet pipeline 4010, the minimum size of the functionalized carrier liquid inlet is 200 μm, the liquid inlet pipeline 307 is other liquid injection ports, including probes and samples, and the minimum size of the port is 80 μm. The liquid outlet pipeline discharges waste liquid, which is shared by all units. Each reaction chamber was a 2mm diameter circular chamber. Every 3 detection units form a detection path, and the detection path can be used as a detection window of the same sample by combining with the functionalized hydrogel particles. Each detection unit can accommodate hundreds of hydrogel particles, so the detection chip can provide detection of at least 120 miRNAs. The chip can provide high-flux and multi-parallel experimental results, and provides multiple possibilities and feasible realizability for detecting miRNA for a complex organ chip system.
The two-stage HCR reaction in the detection chip respectively comprises a first-stage HCR amplification designed for the detection target and based on the HCR amplification principle, and comprises a secondary HCR probe sequence and a HCR amplification sequence thereof. The primary HCR amplification sequences are respectively provided with secondary HCR complementary tail ends, and the H3 sequence of the secondary HCR is respectively complementary with the tail ends of the primary HCR amplification sequences. The amplification sequence of the secondary HCR is general, and only the amplification sequence of the primary HCR needs to be modified aiming at the target to be detected.
As shown in FIG. 15, the HCR reaction principle is that the specifically designed stem-loop probes H1 and H2 can form a stem-loop structure with complementary ends and a middle loop after annealing. When the target miRNA exists, the target miRNA can be hybridized with the 3 '-end of the H1 probe, so that the original stem-loop structure is opened, and the special sticky end of the 5' -end is exposed. The cohesive end can be complementarily hybridized with the 3' -end of the H2 probe, so that the stem-loop structure of H2 is opened. Through cyclic and repeated ring opening and hybridization, a large number of H1 and H2 probes are combined alternately and extended into linear double-stranded DNA products containing repeated units. The functionalized hydrogel is modified by using H1 as a probe through acrydite groups and is subjected to covalent crosslinking with a three-dimensional network skeleton of the hydrogel, so that the hydrogel can be directly fixed on the inner surface and the outer surface of the hydrogel microparticles while being photo-cured, and the functionalization of the microparticles is completed. This patent has designed the detection that two-stage HCR is used for miRNA, when target miRNA participated in, the long dsDNA chain of one-level produced, simultaneously, the sequence tail end of 14nt of H1 and H2 and the complementary pairing of H3 tail end of secondary HCR, and the secondary HCR that adds corresponds the target and induces the amplification reaction of secondary HCR simultaneously. The secondary HCR is unified, so that only the sequence of the primary HCR needs to be designed, which greatly reduces the design challenges for HCR. The two stages combine the advantages of high efficiency, sensitivity and simple operation of isothermal amplification of HCR, and combine the advantages of in-situ detection of fluorescent molecules and low signal to noise ratio, and the combination only needs to provide a proper reaction environment, so that the double signal amplification and detection operation can be completed in a short time.
THP-1 was added to the refreshing transvascular unit model in the upper chamber of the second culture chamber, the same amount of THP-1 was added to the upper chamber of the first culture chamber, and LPS (10. Mu.g/ml) was added to the second culture chamber to construct an encephalitis model as an experimental group, and no LPS was added to the control group. Through the integrated flow path, and according to the steps of the method for using the detection chip, the culture solutions in the first culture chamber and the second culture chamber are injected into the detection chip to detect miRNA-21 and miRNA-let7a, and as a result, as shown in fig. 16, the two miRNAs in the experimental group are found to have a significant increase phenomenon at the blood inlet. This is in combination with Gaudet AD, fonken LK, watkins LR, nelson RJ, popovich PG. MicroRNAs: roles in Regulating neuroi nflash. Neuroscintist. 2018Jun;24 (3) agreement reported in the 221-245 article. The practicability of the detection method and the integrated platform, and the high sensitivity, the strong anti-interference capability and the specificity of the detection method are proved.
The foregoing examples merely illustrate embodiments of the invention and are described in more detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.
Claims (10)
1. The multilayer barrier bionic chip comprises a micro-channel layer, a porous membrane layer, an upper chamber layer and a top cover layer from bottom to top in sequence, and is characterized in that the micro-channel layer and the upper chamber layer share a fluid space and provide a space for cell culture at the lower layer, through holes are formed in the porous membrane layer and the upper chamber layer and are communicated with the micro-channel, the porous membrane layer is provided with a porous membrane with the aperture of 8-15 mu m, the upper chamber layer is provided with a plurality of cell culture chambers, the bottom of each cell culture chamber is provided with a porous membrane of the porous membrane layer, the top cover layer seals the upper chamber layer and seals the through holes of the upper chamber layer, and the micro-channel layer and the upper chamber layer are used for respectively culturing cells at the inner side and the outer side of a simulated key functional interface.
2. The multilayer barrier biomimetic chip of claim 1, wherein the microchannel layer is made of glass, the height of the microchannel is not less than 40 μm, the length of the microchannel corresponds to the upper chamber layer of the chip, the porous membrane layer is formed by clamping a porous membrane by a polydimethylsiloxane PDMS pore plate at the lower part and a glass pore plate at the upper part, the porous membrane is made of a polycarbonate membrane, and the upper chamber layer and the top cover layer are made of polymethyl methacrylate (PMMA).
3. The multilayer barrier biomimetic chip according to claim 1, wherein a culture medium perfusion connector is arranged at a position corresponding to the culture chamber on the top cover layer, and liquid perfusion is performed in the culture chamber through a liquid driving device.
4. The multilayer barrier biomimetic chip according to claim 1, wherein U87 human brain astrocytoma cells are cultured in the microchannel layer, hCMEC/D3 human brain microvascular endothelial cells are cultured in the upper chamber layer in the first culture chamber, SH-SY5Y neuroblastoma cells are cultured in the second culture chamber, and hCMEC/D3 human brain microvascular endothelial cells are cultured in the third culture chamber.
5. A method for constructing a neuro-peripheral immune interaction model using the multi-layer barrier biomimetic chip of claim 1, comprising the steps of:
step 1) sterilizing each structure of the chip, inoculating U87 cells to the micro-channel layer through the through hole of the upper chamber layer, filling the micro-channel layer and the upper chamber layer with a culture medium, covering the chip by using a top cover layer, clamping the top cover layer and the chip for inversion culture until the U87 cells are adhered to the porous membrane on the PDMS pore plate side of the porous membrane layer;
step 2) placing the front surface of the chip, discarding the culture medium, respectively inoculating hCMEC/D3 cells, SH-SY5Y cells and hCMEC/D3 cells to the first cell culture chamber, the second cell culture chamber and the third cell culture chamber of the upper chamber layer, carrying out cell perfusion culture on the first cell culture chamber and the third cell culture chamber after the cells are attached, wherein the perfusion rate is 500 mu L/h, and forming a compact structure by the hCMEC/D3 cells;
step 3) inoculating THP-1 human monocytic leukemia cells into the first cell culture chamber of the upper chamber layer.
6. The method for constructing a neural-peripheral immune interaction model by using the multilayer barrier biomimetic chip according to claim 5, wherein the method comprises the following steps: wherein the cell inoculation number ratio of U87 cells, SH-SY5Y cells, hCMEC/D3 cells and THP-1 cells is (3-10): 1: (5-20): (3-10).
7. An organ chip integrated system comprises an organ chip module, a liquid path control module and a detection chip module, and is characterized in that,
the organ chip module is characterized in that the multilayer barrier bionic chip in claim 3 is placed in a shell, and a temperature control fan and CO are communicated into the shell 2 The liquid inlet liquid paths of the perfusion connectors arranged in the first culture chamber and the third culture chamber of the upper chamber layer are connected with the injection pump, and the liquid outlet liquid paths are respectively connected with the first liquid storage device and the third liquid storage device;
the liquid path control chip of the liquid path control module is connected with the liquid outlet pipelines of the first liquid storage device and the third liquid storage device and the liquid outlet pipeline of the detection reserve liquid, the liquid inlet pipeline of the detection chip is connected with the liquid outlet pipeline of the detection chip, and the liquid path control chip is switched by the injection pump and the gas supply device;
the detection chip module is used for placing the detection chip on the objective table, the led-filtering base is arranged under the objective table, the small microscope is placed on the objective table, the light-shielding shell is arranged outside the objective table, the observation area is arranged on the light-shielding shell, the observation area corresponds to the ocular of the small microscope, the detection chip is connected in series by a plurality of reaction chambers, each reaction chamber is respectively connected with the liquid inlet pipeline, the functionalized carrier liquid inlet and the liquid outlet pipeline, and the liquid outlet pipeline is connected with the liquid storage device.
8. The integrated system of organ chip according to claim 7, wherein the liquid path control chip comprises a substrate layer, a PDMS buffer layer, a gas chamber layer and a liquid path layer from bottom to top, the liquid path layer comprises a plurality of mutually communicated liquid branches, the gas chamber layer is provided with a plurality of gas control units, each liquid branch corresponds to one gas control unit, the gas control units generate gas chamber deformation to control on-off of the liquid branch, the gas supply device uses a pressure control valve to introduce gas in the gas storage bottle into the gas chamber layer, and the electromagnetic valve controls switching of the gas path.
9. The integrated organ-chip system of claim 7, wherein the detection reservoirs are filled with buffer solution, primary HCR amplification probe and secondary HCR priming probe reservoirs, secondary HCR amplification fluorescent probe reservoirs, respectively, and the detection reservoirs are accessed into the fluid circuit control chip by a four-way tube.
10. The integrated system of organ-chip according to claim 9, wherein the liquid inlet pipeline of the detection chip is filled with the culture liquid and the detection reserve liquid flowing out of the first and third liquid storage devices, and the reaction chamber is embedded with a plurality of functionalized hydrogel particles, and the pore diameter of the liquid inlet pipeline of the detection chip is smaller than the particle diameter of the functionalized hydrogel particles.
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