CN114480123A - Integrated microfluidic tissue chip and large-scale stimulus screening and analyzing method - Google Patents

Integrated microfluidic tissue chip and large-scale stimulus screening and analyzing method Download PDF

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CN114480123A
CN114480123A CN202210089735.0A CN202210089735A CN114480123A CN 114480123 A CN114480123 A CN 114480123A CN 202210089735 A CN202210089735 A CN 202210089735A CN 114480123 A CN114480123 A CN 114480123A
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concentration gradient
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stimulus
tissue
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刘文明
孙美林
张晋玮
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Central South University
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Abstract

The invention discloses an integrated microfluidic tissue chip and a large-scale stimulus screening and analyzing method. The chip consists of a flow layer and a control layer. The flowing layer is provided with a liquid inlet, a chemical concentration gradient generator, a micro-cavity and an outlet. The chemical concentration gradient generator is provided with an initial end, a non-irritant unit, a flow distribution and mixing unit, an irritant unit and outlet ends, wherein each initial end is correspondingly connected with a single liquid inlet, and each outlet end is correspondingly connected with a single microcavity; each microcavity is connected to a single outlet. The control layer is provided with an air inlet, a pneumatic micro-structure and an air inlet micro-pipeline with a closed tail end. All the pneumatic microstructures are communicated with the air inlet through a micro pipeline with a closed tail end. The invention can realize the array cell positioning, the large-scale three-dimensional tissue preparation, the large-scale magnitude order concentration gradient type tissue stimulation effect and the analysis thereof, and the controllable recovery of tissue samples with different stimulation in a single chip.

Description

Integrated microfluidic tissue chip and large-scale stimulus screening and analyzing method
Technical Field
The invention relates to the technical field of microfluidic chips, in particular to an integrated microfluidic tissue chip and a large-scale stimulus screening and analyzing method. The method is used for array cell positioning, large-scale three-dimensional tissue preparation, large-scale order of magnitude concentration gradient type tissue stimulation and analysis thereof, and controllable recovery of tissue-like samples with different stimulations.
Background
The in vitro cell culture model is an effective tool in the fields of biomedical basic research, drug research and development and the like. In the traditional two-dimensional cell culture mode, cells are usually cultured in a culture dish or a pore plate, the environment of the cells is far away from the growth environment of the cells in vivo tissues, and the functional expression and metabolic activity of the cells cannot accurately reflect the real in vivo state, so that the effect evaluation of the effectiveness analysis of experimental results, particularly the effect evaluation of drug screening experiments, can be influenced. The three-dimensional structure formed by cell aggregation, also called three-dimensional tissue, can provide the spatial structure and growth environment similar to those of cells in vivo, including cell-to-cell, cell-to-extracellular matrix interaction, signal transmission and the like. Three-dimensional tissues have become powerful tools in the research fields of basic research of life science, tissue engineering, regenerative medicine, cancer, drug research and development and the like, and are receiving wide attention. In recent decades, global researchers have constructed various in vitro three-dimensional tissue-like preparation methods, such as anti-adhesion methods, suspension drop methods, and porous material methods, and used for screening, analysis and evaluation. However, these methods mostly have the disadvantages of complicated steps, time and labor consuming operation, poor geometric uniformity of similar tissues, and the like, and are not conducive to efficient preparation of similar tissues and construction of in vitro three-dimensional models.
The micro-fluidic chip can realize the accurate control of micro-fluid, reduce the labor time and intensity of manual operation, and avoid errors and the like possibly caused by the manual operation. The micro-fluidic chip also has the advantages of low cost, low consumption, high flux, accurate control, real-time dynamic analysis and the like, can be used for in-vitro construction and analysis application of a tissue-like model, and is beneficial to accelerating the processes of life science research, drug screening, clinical transformation and the like. At present, the micro-fluidic technology has successively realized the high-throughput preparation and analysis application of the homogeneous tissue of matter concentration gradient generation and geometry in the chip. Concentration gradients typically occur in the 3-5 order range. However, the large-scale concentration gradient generation, the high-flux preparation of three-dimensional tissue, the large-scale concentration gradient type synchronous stimulation and analysis of the tissue and the controllable recovery of the tissue sample under different stimulation conditions are difficult to realize in the existing microfluidic chip.
Disclosure of Invention
In order to solve the technical problems, the invention provides an integrated microfluidic tissue chip and a large-scale stimulus screening and analyzing method. The method is used for array cell positioning, large-scale three-dimensional tissue preparation, large-scale order of magnitude concentration gradient type tissue stimulation and analysis thereof, and controllable recovery of tissue-like samples with different stimulations.
The technical scheme for solving the technical problems is as follows: the integrated microfluidic tissue chip comprises a flow layer and a control layer, wherein the flow layer is provided with a liquid inlet, a chemical concentration gradient generator, a microcavity and an outlet which are sequentially connected, the control layer is provided with an air inlet, a pneumatic micro-structure and an air inlet micro-pipeline with a closed tail end, the microcavity corresponds to the plurality of pneumatic micro-structures, and all the pneumatic micro-structures are communicated with the air inlet through the micro-pipeline with the closed tail end.
In the integrated microfluidic tissue chip, the chemical concentration gradient generator is provided with 2 starting ends, 1 non-irritant unit, 6-10 flow distribution and mixing units, 1 irritant unit and 8-12 outlet ends, each starting end is correspondingly connected with 1 inlet, and each outlet end is correspondingly connected with 1 microcavity; each microcavity is provided with 1 outlet, one part of the liquid without the irritant and the liquid with the irritant in each flow distribution and mixing unit enters the microcavity, the other part of the liquid enters the next-stage flow distribution and mixing unit, and dilution is carried out according to the flow ratio of the liquid without the irritant to the liquid with the irritant flowing into the flow distribution and mixing unit.
In the integrated microfluidic tissue chip, the flow ratio of the liquid without stimulus to the liquid containing stimulus flowing into each flow distribution and mixing unit of the chemical concentration gradient generator is 1-20, so that the stimulus-containing mixed liquid diluted by 2-21 times is formed.
The flow ratio of the liquid without the stimulus to the liquid containing the stimulus flowing into each flow distribution and mixing unit of the chemical concentration gradient generator is 9, so that the mixed liquid containing the stimulus with 10-fold dilution is formed, and the whole chemical concentration gradient generator forms 7-11 order of magnitude type concentration gradients.
An integrated microfluidic tissue chip for large-scale stimulus screening and analysis method comprises the following steps:
injecting a solution containing anti-cell adhesion molecules from a liquid inlet, and performing anti-cell adhesion modification on the chemical concentration gradient generator and the microcavity surface;
cleaning the chemical concentration gradient generator and the microcavity with fresh cell culture solution;
perfusing a cell suspension through the fluid inlet;
opening the pneumatic microstructure to capture cells;
perfusing a fresh cell culture solution to enable the cells captured in the pneumatic microstructure to self-assemble into a three-dimensional tissue;
pouring the cell culture solution without the stimulus into the chemical concentration gradient generator from an inlet close to one side of the stimulus unit, and simultaneously pouring the cell culture solution with the stimulus into the chemical concentration gradient generator from an inlet close to one side of the stimulus unit; the perfusion flow ratio of the cell culture solution without the stimulus to the cell culture solution with the stimulus is 1-25; stimulators are distributed and mixed through flow to form large-scale quantitative concentration gradients and enter the corresponding micro-cavities from different outlet ends respectively;
and closing the pneumatic microstructure, discharging the three-dimensional tissue samples under the action of different concentrations of stimulators in different microcavities along with liquid flow to different outlets respectively, collecting different samples, and performing chip-outside biological analysis.
The invention has the beneficial technical effects that: the invention can complete array cell positioning, large-scale three-dimensional tissue preparation, large-scale order of magnitude concentration gradient type tissue stimulation and analysis thereof, and controllable recovery of tissue samples with different stimulations in a single chip. Compared with the traditional microfluidic three-dimensional tissue operation and analysis method, the method has a wider range of stimulus concentration and screening analysis range, has better high-throughput operation and analysis effects, simultaneously enables the tissue-like sample positioning and recovery operation under the stimulation condition of specific concentration to be simpler and more efficient, and can be widely applied to screening analysis application related to various in-vitro bionic tissues.
Drawings
FIG. 1 is a schematic structural diagram of the present invention.
FIG. 2 is a schematic diagram of a chemical concentration gradient generator according to the present invention.
FIG. 3 is a schematic diagram of the cell perfusion sample injection in the integrated microfluidic tissue chip according to the present invention.
FIG. 4 is a schematic diagram of cell capture in an integrated microfluidic tissue chip according to the present invention.
FIG. 5 is a schematic diagram of the formation of three-dimensional tissue-like structures by cell self-assembly in the integrated microfluidic tissue-like chip according to the present invention.
Fig. 6 is a schematic diagram of stimulation-based tissue at a specific concentration of stimuli within an integrated microfluidic-based tissue chip according to the present invention.
Fig. 7 is a schematic diagram of recovery of a specific concentration of stimulus-stimulated tissue sample in an integrated microfluidic tissue-like chip according to the present invention.
Detailed Description
The present invention will be described in detail below with reference to embodiments with reference to the attached drawings.
Example 1
An integrated microfluidic chip used in this example was designed and prepared for the laboratory where the applicant was located. The integrated microfluidic chip is prepared from polydimethylsiloxane, and the integrated microfluidic chip is irreversibly sealed on the surface of glass coated with the polydimethylsiloxane.
As shown in fig. 1 and 2, fig. 1 is a schematic structural diagram of the present invention, and the present invention is composed of a flow layer and a control layer. The chip flow layer comprises 2 liquid inlets 1 and 2, 9 outlets 3, 1 chemical concentration gradient generator 4 and 9 microcavities 5. Wherein, 2 starting ends 21 and 22 of the chemical concentration gradient generator, 9 outlet ends 23, 1 non-irritant unit 24, 7 flow distribution and mixing units 25 and 1 irritant unit 26 are respectively connected with 1 liquid inlet and 1 outlet end respectively; each microcavity 5 is connected to 1 outlet. The supporting microcolumns 6 within the microcavity 5 serve to prevent collapse of the microcavity.
The chip control layer comprises 1 air inlet 7, 108 pneumatic microstructures 8 and 1 micro-pipeline 9 with closed ends. The pneumatic microstructures are divided into 9 groups, each microcavity 5 is provided with a group of horseshoe-shaped pneumatic microstructures 8, each group comprises 12 pneumatic microstructures 8, and all the pneumatic microstructures 8 are communicated with the air inlet 7 through the micro pipeline 9 with the closed tail end.
The ratio of the flow rate of the non-irritant-containing liquid to the flow rate of the irritant-containing liquid flowing into each flow rate distribution and mixing unit of the chemical concentration gradient generator 4 in the present invention is 9, so that 10-fold dilution of the irritant-containing mixed liquid can be formed, and 8 number-order concentration gradients, such as 0.0001, 0.001, 0.01, 0.1, 1, 10, 100 and 1000, can be formed in the whole chemical concentration gradient generator 4.
As shown in fig. 3, fig. 3 is a schematic diagram of cell perfusion sample injection in the integrated microfluidic tissue chip of the present invention, in which 31 is a sample inlet of the micro-cavity 5, 32 is a sample outlet of the micro-cavity 5, and 33 is the pneumatic microstructure 8 and 34 in a closed state are cells.
As shown in fig. 4, fig. 4 is a schematic diagram of cell capture in the integrated microfluidic tissue chip of the present invention, wherein 41 is the pneumatic microstructure 8 in the on state, and 42 is the captured cells.
As shown in fig. 5, fig. 5 is a schematic diagram of the three-dimensional tissue-like structure formed by self-assembly of cells in the integrated microfluidic tissue-like chip of the present invention, wherein 51 is the three-dimensional tissue-like structure formed by self-assembly.
As shown in fig. 6, fig. 6 is a schematic diagram of stimulation-based tissue by a specific concentration of stimuli in the integrated microfluidic-based tissue chip of the present invention, wherein 61 is the specific stimulation substance, and 62 is the stimulation-based tissue by the specific concentration of stimuli.
Fig. 7 is a schematic diagram of the recovery of a specific concentration of stimulus-stimulated tissue-like sample within an integrated microfluidic tissue-like chip according to the present invention, as shown in fig. 7, wherein 71 is the tissue-like sample released and leaving the pneumatic microstructure.
The invention can simultaneously realize the cell positioning capture of 108 sites in the micro-cavity and the precise control of 108 three-dimensional tissues.
When no external air pressure is applied, the horseshoe-shaped pneumatic microstructure 8 is in a closed state and is of a two-dimensional structure; when external air pressure is applied, the pneumatic micro-structure 8 is in an open state, expands and presents a three-dimensional structure in the corresponding micro-cavity, and can be used for finishing cell capture and positioning operation of three-dimensional tissues in the micro-cavity.
The invention can complete the precise control operation and the screening analysis of the large-range concentration gradient type stimulation treatment of the three-dimensional tissue in a single chip.
The invention can realize the controllable recovery of three-dimensional tissue samples processed by stimulators with different concentrations.
Example 2
This example provides a large scale stimulus screening assay for example 1 integrated microfluidic tissue-like chips.
Firstly, injecting a 2mg/mL bovine serum albumin solution from liquid inlets (1 and 2) of a chip flow layer by using a micro-injection pump at the flow rate of 10 mu L/min, allowing the bovine serum albumin to enter all micro-pipelines and micro-cavities in the chip flow layer, incubating for 3 hours at 37 ℃, and performing anti-cell adhesion modification on the inner surfaces of all the micro-pipelines and micro-cavities;
then, filling a fresh cell culture solution, and cleaning all micro-pipelines and micro-cavities in the flow layer of the chip;
the cell density is 5X 105The suspension of the neural stem cells per mL is filled into the microcavity through the liquid inlets 1 and 2, as shown in FIG. 3;
and applying a certain air pressure to the pneumatic microstructure, and starting the pneumatic microstructure, as shown in fig. 4, wherein 41 is the pneumatic microstructure in an open state, 42 is the captured cell, and the neural stem cell is captured and positioned at the microcavity position of the flow layer corresponding to the pneumatic microstructure.
The chip was placed at 37 ℃ in 5% CO2Continuously culturing for 15 days under the condition of saturated humidity to enable the neural stem cells to self-assemble to form a three-dimensional neural groupThe structure is shown in FIG. 5, wherein 51 is a three-dimensional type structure formed by self-assembly.
The cell culture fluid without nerve growth factor is perfused into the chemical concentration gradient generator from the fluid inlet 1 near the side of the non-irritant unit 24 at a perfusion flow rate of 0.71 μ L/min, while the cell culture fluid with nerve growth factor of 100ng/mL is perfused into the chemical concentration gradient generator from the fluid inlet 2 near the side of the irritant unit 26 at a perfusion flow rate of 0.1 μ L/min. Through flow distribution and mixing, 1 blank control solution without nerve growth factor and 8 orders of magnitude nerve growth factor concentration gradient solutions are respectively formed at different outlet ends 23 of the chemical concentration gradient generator, the concentrations of the blank control solutions are respectively 0ng/mL, 0.00001ng/mL, 0.0001ng/mL, 0.001ng/mL, 0.01ng/mL, 0.1ng/mL, 1ng/mL, 10ng/mL and 100ng/mL from left to right, and the blank control solutions and the nerve growth factor concentration gradient solutions respectively enter the corresponding microcavities from different outlet ends.
The nerve-like tissues of different microcavities are affected by nerve growth factors with different concentrations, and then the optical microscopic image acquisition and analysis system is used for carrying out morphological geometrical observation and analysis on the nerve-like tissues under the condition of the action of the nerve growth factors with different concentrations, as shown in fig. 6, 61 is a specific stimulating substance, and 62 is a tissue-like tissue stimulated by the stimulus with specific concentration.
When nerve-like tissue samples processed by nerve growth factors with different concentrations need to be recovered, the pneumatic microstructure is closed, the three-dimensional nerve-like tissue samples under the action conditions of the nerve growth factors with different concentrations in different microcavities are discharged along with liquid flow, as shown in fig. 7, wherein 71 is the nerve-like tissue samples released and leaving the pneumatic microstructure, and respectively reach different outlets, so that the recovery operation of different samples is completed. The recovered sample can be subjected to off-chip biological analysis, such as ultrastructural analysis of a neuroid tissue sample using an electron microscope.
Example 3
This example provides a large scale stimulus screening assay for example 1 integrated microfluidic tissue-like chips.
Firstly, injecting a polyethylene glycol solution with the concentration of 1mg/mL from liquid inlets (1 and 2) of a chip flow layer by using a micro-injection pump at the flow rate of 15 mu L/min, allowing the polyethylene glycol to enter all micro-pipelines and micro-cavities in the chip flow layer, incubating at room temperature for 2 hours, and performing anti-cell adhesion modification on the inner surfaces of all the micro-pipelines and micro-cavities;
then, filling a fresh cell culture solution, and cleaning all micro-pipelines and micro-cavities in the flow layer of the chip;
the cell density was 1X 106The individual/mL of the hepatocyte suspension is filled into the microcavity through the liquid inlets 1 and 2;
and applying a certain air pressure (20psi) to the pneumatic microstructure, opening the pneumatic microstructure, and capturing and positioning the hepatic cells at the microcavity position of the flow layer corresponding to the pneumatic microstructure.
The chip was placed at 37 ℃ in 5% CO2And continuously culturing for 6 days under the saturated humidity condition to enable the hepatocyte to self-assemble to form the three-dimensional liver-like tissue.
Cell culture fluid without rifampicin was perfused into the chemical concentration gradient generator from the fluid inlet 1 near the side of the non-irritant unit 24 at a perfusion flow rate of 14.2. mu.L/min, while cell culture fluid with rifampicin of 1000. mu.M was perfused into the chemical concentration gradient generator from the fluid inlet 2 near the side of the irritant unit 26 at a perfusion flow rate of 2. mu.L/min. Through flow distribution and mixing, 1 blank solution without rifampicin and 8 orders of magnitude rifampicin concentration gradient solutions are formed at different outlet ends 23 of the chemical concentration gradient generator, respectively, and the concentrations thereof are 0 μ M, 0.0001 μ M, 0.001 μ M, 0.01 μ M, 0.1 μ M, 1 μ M, 10 μ M, 100 μ M, 1000 μ M from left to right, and enter the corresponding micro-cavities from different outlet ends, respectively.
The liver-like tissues of different microcavities are affected by rifampicin with different concentrations, and then the activity of the liver-like tissues under the condition of rifampicin with different concentrations is analyzed by using a fluorescent staining method and an optical microscopic image acquisition and analysis system.
When the hepatic tissue samples treated by rifampicin with different concentrations need to be recovered, the pneumatic microstructure is closed, the three-dimensional hepatic tissue samples under the action conditions of rifampicin with different concentrations in different micro cavities are discharged along with liquid flow and respectively reach different outlets, and thus the recovery operation of different samples is completed. The recovered heparinoid tissue samples may be subjected to off-chip biological analysis, such as quantitative analysis of the heparinoid tissue samples using a flow cytometer.
Example 4
This example provides a large scale stimulus screening assay for example 1 integrated microfluidic tissue-like chips.
Firstly, injecting Pluronic F127 solution with the concentration of 6mg/mL from liquid inlets 1 and 2 of a chip flow layer by using a micro-injection pump at the flow rate of 12 mu L/min, allowing the Pluronic F127 to enter all micro-channels and micro-cavities in the chip flow layer, incubating at room temperature for 3 hours, and performing anti-cell adhesion modification on the inner surfaces of all the micro-channels and micro-cavities;
then, filling a fresh cell culture solution, and cleaning all micro-pipelines and micro-cavities in the flow layer of the chip;
the cell density is 2X 106one/mL of breast cancer cell suspension is perfused into the microcavity by fluid inlets 1 and 2, as shown in fig. 3;
and applying a certain air pressure effect on the pneumatic microstructure, starting the pneumatic microstructure, and capturing and positioning the breast cancer cells at the microcavity position of the flow layer corresponding to the pneumatic microstructure.
The chip was placed at 37 ℃ in 5% CO2And continuously culturing for 5 days under the saturated humidity condition to enable the breast cancer cells to self-assemble into a three-dimensional breast cancer-like tissue, as shown in figure 5.
Cell culture fluid without gemcitabine is perfused into the chemical concentration gradient generator from fluid inlet 1 near the side of the no-stimulus unit 24 at a perfusion flow rate of 7.1 μ L/min, while cell culture fluid with gemcitabine at a concentration of 1000 μ g/mL is perfused into the chemical concentration gradient generator from fluid inlet 2 near the side of the stimulus unit 26 at a perfusion flow rate of 1 μ L/min. Through flow distribution and mixing, 1 blank solution without gemcitabine and 8 magnitude gemcitabine concentration gradient solutions are formed at different outlet ends 23 of the chemical concentration gradient generator, and the concentrations of the blank solutions are respectively 0 mug/mL, 0.0001 mug/mL, 0.001 mug/mL, 0.01 mug/mL, 0.1 mug/mL, 1 mug/mL, 10 mug/mL, 100 mug/mL and 1000 mug/mL from left to right, and the blank solutions enter the corresponding microcavity diagram 1 from different outlet ends respectively.
The breast cancer-like tissues of different microcavities are affected by gemcitabine with different concentrations, and then the breast cancer-like tissues under the condition of different concentrations of gemcitabine are subjected to activity and apoptosis analysis by using a fluorescence staining method and an optical microscope image acquisition and analysis system.
When the breast cancer-like tissue samples treated by gemcitabine with different concentrations need to be recovered, the pneumatic microstructure is closed, the three-dimensional breast cancer-like tissue samples 71 under the action conditions of gemcitabine with different concentrations in different micro cavities are discharged along with liquid flow and respectively reach different outlets, and thus, the recovery operation of different samples is completed. The recovered breast cancer-like tissue sample can be subjected to off-chip biological analysis, such as immunohistochemical analysis based on sectioning techniques.

Claims (10)

1. The integrated microfluidic tissue chip is characterized by comprising a flow layer and a control layer from top to bottom, wherein the flow layer is provided with a liquid inlet, a chemical concentration gradient generator, a microcavity and an outlet which are sequentially connected, the control layer is provided with an air inlet, a pneumatic micro-structure and an air inlet micro-pipeline with a closed tail end, the microcavity corresponds to the plurality of pneumatic micro-structures, and all the pneumatic micro-structures are communicated with the air inlet through the micro-pipeline with the closed tail end.
2. The integrated microfluidic-like tissue chip of claim 1, wherein: the chemical concentration gradient generator is provided with 2 starting ends, 1 irritant unit, 6-10 flow distribution and mixing units, 1 irritant unit and 8-12 outlet ends, wherein each starting end is correspondingly connected with 1 liquid inlet, and each outlet end is correspondingly connected with 1 microcavity; each microcavity is provided with 1 outlet, one part of the liquid without the irritant and the liquid with the irritant in each flow distribution and mixing unit enters the microcavity, the other part of the liquid enters the next-stage flow distribution and mixing unit, and dilution is carried out according to the flow ratio of the liquid without the irritant to the liquid with the irritant flowing into the flow distribution and mixing unit.
3. The integrated microfluidic tissue chip of claim 2, wherein: the flow ratio of the liquid without the irritant to the liquid with the irritant which flows into each flow distribution and mixing unit of the chemical concentration gradient generator is 1-20, and the mixed liquid with the irritant with 2-21 times of dilution is formed.
4. The integrated microfluidic tissue chip according to claim 3, wherein: the flow ratio of the liquid without the stimulus to the liquid containing the stimulus flowing into each flow distribution and mixing unit of the chemical concentration gradient generator is 9, so that the mixed liquid containing the stimulus with 10-fold dilution is formed, and the whole chemical concentration gradient generator forms 7-11 order of magnitude type concentration gradients.
5. The integrated microfluidic-like tissue chip of claim 1, wherein: each microcavity corresponds to 10-35 pneumatic microstructures.
6. The integrated microfluidic-like tissue chip of claim 1, wherein: the integrated microfluidic tissue chip is made of polydimethylsiloxane or an elastic high polymer material.
7. The integrated microfluidic tissue chip of claims 1-6 for large-scale stimulus screening analysis, comprising the steps of:
a solution containing anti-cell adhesion molecules is poured from a liquid inlet, and anti-cell adhesion modification is carried out on the chemical concentration gradient generator and the microcavity surface;
cleaning the chemical concentration gradient generator and the microcavity with fresh cell culture solution;
perfusing a cell suspension through the fluid inlet;
opening the pneumatic microstructure to capture cells;
perfusing a fresh cell culture solution to enable the cells captured in the pneumatic microstructure to self-assemble into a three-dimensional tissue;
pouring the cell culture solution without the stimulus into the chemical concentration gradient generator from an inlet close to one side of the stimulator unit, and simultaneously pouring the cell culture solution with the stimulus into the chemical concentration gradient generator from an inlet close to one side of the stimulator unit; the perfusion flow ratio of the cell culture solution without the stimulus to the cell culture solution with the stimulus is 1-25; stimulators are distributed and mixed through flow to form large-scale quantitative concentration gradients and enter the corresponding micro-cavities from different outlet ends respectively;
and closing the pneumatic microstructure, discharging the three-dimensional tissue samples under the action of different concentrations of stimulators in different microcavities along with liquid flow to different outlets respectively, collecting different samples, and performing chip-outside biological analysis.
8. The large scale stimulus screening assay of claim 7, wherein: the anti-cell adhesion molecule is Pluronic F-127, bovine serum albumin or polyethylene glycol.
9. The method of claim 7, wherein: the cell is a primary cell or a cell line of a mammal.
10. The method of claim 7, wherein: the stimulant is various natural product molecules, artificial synthetic molecules, cytokines, growth factors, proteins or nucleic acids.
CN202210089735.0A 2022-01-25 2022-01-25 Integrated microfluidic tissue chip and large-scale stimulus screening and analyzing method Pending CN114480123A (en)

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