CN114231397B - Centrifugal microfluidic system for high-flux drug testing and application method thereof - Google Patents
Centrifugal microfluidic system for high-flux drug testing and application method thereof Download PDFInfo
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- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
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- C—CHEMISTRY; METALLURGY
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- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
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Abstract
The invention belongs to the technical field of microfluidic chips, and particularly relates to a centrifugal microfluidic system for testing high-flux drugs and a use method thereof. Can realize three-dimensional culture of single-kind cells with high flux and micro-quantization or three-dimensional co-culture of multicellular indirect contact type and drug test. Comprises a chip; the chip is used for three-dimensional culture of single-kind cells and high-flux drug test; the chip comprises: a top layer, a middle layer and a bottom layer; the top layer is provided with gel fluid injection holes and microgel crosslinking reagent injection holes distributed in a surrounding manner and drug injection holes; the middle layer is provided with a surrounding type concentration gradient forming channel, a microgel collecting liquid pool through hole and a gel fluid channel, the gel fluid channel is communicated with the liquid pool through hole, and a concentration gradient outlet of the concentration gradient forming channel is communicated with the liquid pool through hole; the drug injection hole is communicated with the middle layer hole of the middle layer; the bottom layer is a flat plate layer, and the three PDMS layers are connected in a bonding and sealing way.
Description
Technical Field
The invention belongs to the technical field of microfluidic chips, and particularly relates to a centrifugal microfluidic system for testing high-flux drugs and a use method thereof.
Background
With the continuous and deep research of tumors, higher requirements are also put forward on a cell level drug testing platform. Firstly, a great number of researches show that in a three-dimensional culture environment and a co-culture environment of other related stromal cells, a drug test result has obvious difference from a two-dimensional environment for independently culturing the tumor cells, namely, the response of the tumor cells to the drug is closely related to a tumor microenvironment, so that the simulation of the tumor microenvironment is a key for acquiring response information of a near clinical drug. Meanwhile, different medicines are reasonably combined to play a synergistic effect, and the traditional Chinese medicine composition has a better treatment effect compared with single medicine. However, the combination drug needs to find a proper drug combination and concentration ratio to determine the optimal drug combination condition, and the reliability of the scheme is determined through a plurality of parallel experiments, so that an automatic, integrated and high-throughput test technology is needed. In addition, the characteristic of cell quantity micro-quantization in the drug test platform can obviously reduce the drug test cost. In combination with analysis, the development of an automatic, integrated, micro-quantitative and high-flux cell level drug testing platform based on tumor microenvironment is of great significance to drug development and clinical drug guidance.
The microfluidic chip has the advantages of simple operation, high flux and low cost, and particularly has the advantage of integrating a plurality of functional units and analysis processes into a whole for continuous operation. The droplet microfluidic is a technology for manipulating tiny volume liquid developed based on the microfluidic technology, can generate a large number of cell micro-culture chambers with uniform size and micron level size in a short time, has advantages in aspects of high flux, micro-quantization and the like compared with the conventional microfluidic technology, and has been used for constructing a drug screening platform.
However, at present, based on conventional microfluidic technology and droplet microfluidic technology, a three-dimensional co-culture model of various relevant cells such as tumor cells and relevant stromal cells is constructed with high throughput, and meanwhile, the research of a drug testing platform integrating the automatic configuration of a drug concentration gradient generating unit and the addition of drugs is still in a blank stage. The construction of the research platform enables the medicine test result to reflect the in-vivo situation more truly, is convenient for analyzing the effect of different cells and medicines with different concentration ratios in an interaction mode, has the advantages of high flux and microminiaturization, and has wide application prospect in medicine research and development and clinical medicine guiding.
Disclosure of Invention
The invention provides a centrifugal microfluidic system for testing high-flux drugs and a use method thereof, aiming at the defects in the prior art. The single-phase microgel and the multiphase microgel which are prepared by utilizing the high flux of the centrifugal chip are used as independent three-dimensional culture brackets of tumor cells or non-direct contact three-dimensional co-culture brackets of tumor cells and related stromal cells, and a surrounding type concentration gradient generating unit is designed to perform the perfusion effect of concentration gradient multicomponent drugs on the tumor culture model based on gel particles, so that the centrifugal microfluidic chip capable of realizing the three-dimensional culture of single-kind cells or the non-direct contact three-dimensional co-culture of multicellular and drug testing with high flux and micro-quantization is provided, and the application of the microfluidic chip is provided.
The invention aims to solve the technical problems: the method has the advantages of high throughput, automation, microminiaturization and simulation of tumor microenvironment, and fills the research blank of high throughput drug testing by combining the gel particles formed by the droplet microfluidic with the concentration gradient forming unit.
In order to achieve the above purpose, the present invention adopts the following technical scheme.
The centrifugal microfluidic system for high-flux drug testing comprises a chip; the chip is characterized in that the chip is used for three-dimensional culture of single-kind cells and high-flux drug test; the chip comprises three layers of PDMS: a top layer, a middle layer and a bottom layer; the top layer is provided with gel fluid injection holes and microgel crosslinking reagent injection holes distributed in a surrounding manner and drug injection holes; the middle layer is provided with a surrounding type concentration gradient forming channel, a microgel collecting liquid pool through hole and a gel fluid channel which uniformly radiates outwards, the gel fluid channel is communicated with the liquid pool through hole, and a concentration gradient outlet of the concentration gradient forming channel is communicated with the liquid pool through hole; the drug injection hole is communicated with the middle layer hole of the middle layer; the bottom layer is a flat plate layer, and the three PDMS layers are connected in a bonding and sealing way.
Further, the three layers of PDMS are concentric circles and have the same diameter.
Further, a high-throughput drug testing centrifugal microfluidic system comprises a chip; the chip is characterized in that the chip is used for three-dimensional culture of two cells or three cells and high-flux drug test;
the chip comprises three layers of PDMS: a top layer, a middle layer and a bottom layer; the top layer is provided with gel fluid injection holes and microgel crosslinking reagent injection holes distributed in a surrounding manner and drug injection holes; the middle layer is provided with a surrounding type concentration gradient forming channel, a microgel collecting liquid pool through hole and a gel fluid channel which uniformly radiates outwards, the gel fluid channel is communicated with the liquid pool through hole, and a concentration gradient outlet of the concentration gradient forming channel is communicated with the liquid pool through hole; the drug injection hole is communicated with the middle layer hole of the middle layer; the bottom layer is a flat plate layer; the three PDMS layers are bonded and connected;
wherein, for three-dimensional culture of two cells and high-throughput drug testing, the gel fluid channel comprises a plurality of closely adjacent gel channel groups distributed in groups; each channel group consists of two adjacent gel channels;
wherein, for three-dimensional culture of three cells and high-throughput drug testing, the gel fluid channel comprises a plurality of closely adjacent gel channel groups distributed in groups; each channel group consists of three immediately adjacent gel channels.
Further, the concentration gradient forming channel is formed by a plurality of circumferentially distributed liquid separating ports, liquid collecting ports and serpentine mixing channels step by step.
Furthermore, the chip comprises three or more liquid pools which are mutually connected in series and outlets for forming concentration gradients, so that continuous infusion and drug stimulation of a plurality of concentration gradient drugs can be realized.
Further, when the chip rotates at a high speed, the gel fluid in the chip channel is broken by centrifugal force against surface tension to form independent liquid drops from the outlet, and the independent liquid drops are crosslinked with the gel crosslinking reagent CaCl in the liquid pool 2 Cross-linking polymerization to form single-phase microgel and/or double-phase microgel and/or three-phase microgel.
The application method of the centrifugal microfluidic system for testing the high-flux medicine is characterized by comprising the following steps of: step 1: and (2) packaging cells based on gel particles, and testing medicaments.
Further, in step 1, it includes:
step 1.1, dispersing target cells in sodium alginate gel, introducing the gel into a syringe, pushing the syringe by using a microinjection pump to inject gel fluid of the dispersed cells into a gel fluid channel, and using the microinjection pump to gel CaCl of sodium alginate 2 Injecting into a liquid pool; fixing the centrifugal chip on a spin coaterObtaining gel particles for encapsulating cells on a heart turntable;
step 1.2, caCl in the liquid pool 2 The culture medium was replaced with fresh medium.
Further, the centrifugal revolution number in the formation process of the liquid drop gel particles is 500-5000 r/min (adjusted according to the particle size and the grid density of the target gel particles) for 0.5-2min.
Further, in step 2, it includes:
step 2.1, respectively introducing the same medicine into the two medicine injection holes at the same pushing speed through a microinjection pump; the concentration gradient structure unit is used for obtaining the concentration of a plurality of concentration gradients of one drug or the concentration of two combined drugs with different proportions, the drug continuously enters the liquid pool to react with cells in gel particles, and the liquid outlet of the liquid pool keeps stable liquid flow in the liquid pool;
and 2.2, evaluating the effects of different gradient drugs on cells and the expression conditions of related proteins through dead and alive staining and immunofluorescence experiments, and screening the medication scheme.
Compared with the prior art, the invention has the beneficial effects.
The centrifugal microfluidic system constructed by the invention generates microgels with different structures of single phase, double phase and three phase through centrifugal force driving, and constructs a tumor three-dimensional culture and related stromal cell three-dimensional co-culture system.
The invention designs the surrounding type concentration gradient generating unit aiming at the disc type structure of the centrifugal chip, and automatically configures and adds medicines at the periphery of the cell culture pond, so that the medicine perfusion culture and the construction of tumor microenvironment lead the medicine test result to reflect the in-vivo situation more truly, and the invention is convenient for analyzing the actions of different cells and medicines with different concentration ratios under the interaction mode.
Drawings
The invention is further described below with reference to the drawings and the detailed description. The scope of the present invention is not limited to the following description.
FIG. 1 is a top view of a centrifugal microfluidic chip for three-dimensional cell culture and high throughput drug testing based on monophasic gel particles according to the present invention.
Fig. 2 is a schematic diagram of the structure of each layer of a centrifugal microfluidic chip for three-dimensional cell culture and high-throughput drug testing based on single-phase gel particles.
FIG. 3a is a top view of a centrifugal microfluidic chip of the present invention for three-dimensional co-culture of two cells and high throughput drug testing based on biphasic gel particles; fig. 3b is a top view of a centrifugal microfluidic chip of the invention for three-dimensional co-culture of three cells and high throughput drug testing based on three-phase gel microparticles.
FIG. 4 is a partial schematic view of a concentration gradient forming unit of the present invention.
FIG. 5 is a schematic diagram of single phase gel particles, two phase gel particles and three phase gel particles generated by centrifuging a microfluidic chip.
FIG. 6 shows that the LoVo cells of colorectal cancer in example 1 can normally grow and proliferate in monophasic gel particles.
Detailed Description
As shown in fig. 1-6, the present invention comprises a centrifugal microfluidic chip (fig. 1) for three-dimensional culture and high-throughput drug testing of single cell types based on gel particles, wherein the chip is composed of three layers of PDMS, namely a top layer, a middle layer and a bottom layer; the three layers of PDMS are concentric circles and are equal in size; the top layer PDMS (figure 2 a) is provided with a gel fluid injection hole 1, a microgel crosslinking reagent injection hole 2 and a medicine injection hole 3, wherein the microgel crosslinking reagent injection hole is also a liquid outlet hole 3 in the medicine injection stage; the middle layer substrate (figure 2 b) is sealed by a channel layer and a substrate layer, and then is perforated to obtain a collecting liquid pool through hole, wherein the collecting liquid pool through hole comprises a surrounding type concentration gradient forming channel 4, a microgel collecting liquid pool through hole 5 and a gel fluid channel 6 which uniformly radiates outwards, the gel fluid channel is communicated with the liquid pool, a concentration gradient outlet 7 of the concentration gradient forming channel is communicated with the liquid pool, and a top layer liquid injection hole 3 is communicated with a middle layer hole b; the bottom PDMS is a flat plate layer (fig. 2 c). The multi-layer structure of the chip enables the gel particles to keep a certain distance from the upper wall and the lower wall of the chip in the forming process, and deformation or fusion caused by collision is effectively prevented.
Description of working principle: sodium alginate gel fluid enters the chip from gel fluid injection holes 1 in the top layer substrate a and flows to gel fluid channels 6 of the middle layer substrate b; the microgel crosslinking reagent enters the chip from the microgel crosslinking reagent injection hole 2 in the top layer substrate a, flows to the liquid pool through hole 5 in the middle layer substrate b, and is sealed with the bottom layer substrate c because the liquid pool through hole, namely, the microgel crosslinking reagent enters the liquid pool. After the reagent enters the chip, the centrifugal force is driven, the gel fluid is broken by the centrifugal force against the surface tension to form independent liquid drops from the outlet, and the independent liquid drops and the microgel crosslinking reagent in the liquid pool are subjected to gel crosslinking to form microgel. The three fluid injection holes in the top layer substrate a are gel fluid injection holes 1, microgel crosslinking reagent injection holes 2 and medicine injection holes 3, the injection holes in the top layer substrate a are all communicated with the middle layer substrate b in the vertical direction, and the fluid introduced from the top layer substrate a is respectively introduced into a gel fluid channel 6, a liquid pool through hole 5 and a concentration gradient forming channel 4 through the communicated holes. The gel fluid can be doped into cells, gel particles for encapsulating the cells can be realized by the same principle, and further the drug test can be performed through the concentration gradient forming channel 4. The high-concentration medicine and the low-concentration medicine respectively enter the chip from two medicine injection holes 3 in the top layer substrate a and flow to a concentration gradient forming channel 4 in the middle layer substrate b, pass through a surrounding Christmas tree-shaped concentration gradient forming channel, form a plurality of groups of medicine concentrations and respectively flow into the liquid pool through a concentration gradient outlet of the concentration gradient forming channel, and then perform medicine action with microgel of encapsulated cells in the liquid pool, so that the medicine test based on gel particles is realized at high flux.
The invention is based on a centrifugal microfluidic chip which can be used for non-contact three-dimensional co-culture of two cells (figure 3 a) or three cells (figure 3 b) and high-flux drug testing, and the periphery of the chip also comprises a surrounding type concentration gradient forming channel 8. Such co-culture chips are characterized in that several sets (three sets or more) of circumferentially distributed gel fluid injection ports and immediately adjacent gel fluid channels for generating biphasic or triphasic gel particles are designed, respectively, wherein the chip for generating biphasic gel particles is a plurality of two immediately adjacent gel channels 9 distributed in groups, and the chip for generating triphasic gel particles is a plurality of three immediately adjacent gel channels 10 distributed in groups.
The surrounding type concentration gradient forming channels 4 and 8 are formed by a plurality of circumferentially distributed liquid separating ports 11, liquid collecting ports 12 and serpentine mixing channels 13 step by step (figure 4), the width of the concentration gradient forming channels is 20-200 microns, and the concentration gradient forming device is suitable for constructing concentration gradient forming units on the periphery of a centrifugal type equal-annular chip structure. The mixing channel is designed as a serpentine channel, which aims to quickly realize liquid mixing with different concentrations and save the occupied area of the channel in a chip, but is not limited to the serpentine mixing channel shown in the figure, and can also form channels for other surrounding concentration gradients which can realize mixing of two liquids. The number of concentration gradients formed is consistent with the number of liquid pools connected in series. The surrounding type concentration gradient forming unit structure is the same as the Christmas tree structure concentration gradient forming structure principle of the traditional microfluidic forming concentration gradient, and according to the laminar flow principle, the initial two fluids are repeatedly split, mixed and converged through the micro-channels to form a discontinuous concentration gradient.
The chip comprises three or more liquid pools which are mutually connected in series and outlets for forming concentration gradients, so that continuous infusion and drug stimulation of a plurality of concentration gradient drugs can be realized. For example, as shown in fig. 1, the chip includes 12 liquid pools, the concentration gradient channels can obtain 12 concentration gradient drugs, and the concentration gradient channels, the concentration gradient outlets and the liquid pools are mutually connected in series to form 12 groups of test platforms which can continuously perform concentration gradient drug actions on cells in the microgel.
When the chip rotates at high speed, the gel fluid in the chip channel is subjected to centrifugal force to overcome the surface tension and break off from the outlet to form independent liquid drops (figure 5) which are crosslinked with the gel crosslinking reagent CaCl in the liquid pool 2 Cross-linking polymerization to form a plurality of single-phase microgels 14, 15 and 16, wherein the single-phase microgels can not only encapsulate a single kind of cells but also be used for a plurality of kinds of cellsCo-culture of cells without zoning. When the centrifugal chip rotates at a high speed, the fluid stress in each channel tends to be consistent, so that the microgel has better size uniformity and preparation reproducibility. The width of each outlet of the gel channel is 30-100 micrometers, and the width of the outlet of the drug concentration gradient connecting channel connected with the liquid pool in series is 40-80 micrometers.
The centrifugal microfluidic drug testing platform provided by the invention is established according to the following method:
and (3) manufacturing a chip: the chip positive film is manufactured by designing chip configuration, printing and manufacturing mask, spin coating photoresist on a silicon chip, pre-baking, post-baking, exposing, developing and hardening. And pouring PDMS on the positive film, removing the film layer containing the channels after heating and curing, sealing with the substrate layer, and punching to obtain a collecting liquid pool through hole 5, thereby completing the preparation of the chip intermediate layer b. And then sealing the middle layer with the top layer a, punching, and finally sealing with the bottom substrate layer c to finish the chip manufacture. The chip is sterilized and then used.
Encapsulation of cells based on gel microparticles: dispersing target cells in sodium alginate gel, introducing into a syringe, pushing the syringe by using a microinjection pump to inject gel fluid of dispersed cells into a gel fluid channel, and using the microinjection pump to gel CaCl of sodium alginate 2 Injecting into a liquid pool; and fixing the centrifugal chip on a centrifugal turntable of a spin coater, wherein the centrifugal revolution of the droplet gel particles in the formation process is 500-5000 r/min (regulated according to the particle size and the grid density of the target gel particles) for 0.5-2min, and obtaining the gel particles for encapsulating cells. CaCl in the liquid pool 2 The culture medium was replaced with fresh medium.
Drug testing: the same drug high-concentration solution and low-concentration solution or the two drugs are respectively introduced into the two drug injection holes through a microinjection pump at the same pushing speed. Through the concentration gradient structure unit, the concentration gradient of one medicine or the concentration of two combined medicines in different proportions is obtained, the medicine continuously enters the liquid pool to react with cells in gel particles, and the liquid flow in the liquid pool is kept stable by the liquid outlet of the liquid pool. The effect of different gradient drugs on cells and the expression condition of related proteins are evaluated through dead-living staining and immunofluorescence experiments, and an optimal drug use scheme is screened out, so that the purposes of high-flux and automatic drug testing are achieved.
Specific experimental example 1: obtaining tumor cell three-dimensional culture microgel based on centrifugal microfluidic chip and testing the drug.
The chip is formed by bonding and sealing a plurality of PDMS layers, the internal structure is shown in figure 2, the preparation process is shown in claim 1, and the chip is sterilized under high pressure for standby. The three-dimensional culture microgel of tumor cells based on the chip is obtained as follows: firstly, 0.5mL (1.6% w/v) of sodium alginate gel fluid is added with 0.05mL of cell suspension, the mixture is uniformly mixed and injected into a gel fluid channel, and then 1.5% w/v concentration calcium chloride aqueous solution is injected into a liquid pool. Then, the chip was placed on a centrifugal turntable (KW-4A type desktop spin coater of China academy of sciences microelectronics research institute), and centrifuged at 4200rpm for 1min to obtain gel droplets of encapsulated cells, and the gel droplets were gelled in a liquid bath containing calcium chloride aqueous solution to form gel particles. After the microgel is obtained, sucking the calcium chloride aqueous solution, and cleaning the microgel by using a culture medium containing 10% of serum, thereby obtaining the high-flux microgel which can be used for cell culture and drug testing. The microgels in different liquid pools obtained by the conditions have satisfactory morphology and monodispersity, and the single centrifugation in a single liquid pool can obtain a large amount of microgels, which shows the advantage of high flux of the method. Meanwhile, cells in the obtained microgel are subjected to dead and alive staining, a living cell dye Calcein AM is diluted by a serum-free culture medium in a volume ratio of 1:2000, a dead cell dye Ethidium Homodimer (EthD-1) is diluted by a serum-free culture medium in a volume ratio of 1:500, then two dye dilutions are mixed in a volume ratio of 1:1, the mixed dye is introduced into a liquid pool to incubate with the cells in the microgel for 30min at 37 ℃, and the dead and alive staining condition of the cells is observed under a fluorescent inverted microscope and photographed. The results show that the cells are encapsulated by microgel without affecting the cell activity.
In order to prove that cells can grow and proliferate normally in the microgel so as to meet the requirement of subsequent drug tests, the cells in the microgel obtained by the method are cultured and the growth condition of the cells in the gel is judged by dead and alive dyeing. And culturing and dead-living staining cells in the obtained microgel by taking the colorectal cancer LoVo cells as model cells. The result of cell death and living staining shows that the cells can keep high activity in the microgel for a long time, and the cells are more dense in the gel compared with the result of the first day, so that proliferation activity is shown, and the basic requirements of subsequent experiments such as drug testing are met (figure 6).
Injecting a cell culture medium containing 80mM 5-fluorouracil and a culture medium without a drug into a chip concentration gradient unit respectively, automatically configuring and adding the drug by using the concentration gradient forming unit, performing drug testing on cells in microgels in a liquid pool, performing continuous perfusion type drug stimulation on gel particles containing LoVo cells in the liquid pool, performing dead and alive staining on the cells in the gel after 24 hours of drug stimulation, and evaluating the drug testing result. The 5-fluorouracil with the concentration gradient of 80mM to 0mM has obvious drug effect after the effect of cells, and shows the concentration-effect relationship, so that the chip can be used for high-flux drug testing based on three-dimensional culture of tumor cells.
The invention designs the surrounding type concentration gradient generating unit aiming at the disc type structure of the centrifugal chip, and automatically configures and adds medicines at the periphery of the cell culture pond, so that the medicine perfusion culture and the construction of tumor microenvironment lead the medicine test result to reflect the in-vivo situation more truly, and the invention is convenient for analyzing the actions of different cells and medicines with different concentration ratios under the interaction mode.
The invention establishes a droplet microfluidic high-flux drug testing system integrating a concentration gradient forming unit, gel particles formed by a droplet microfluidic technology can simulate the true three-dimensional microenvironment of tumor cell growth to a certain extent, and provides a new technical platform for drug testing, and has research value and economic value. Meanwhile, the characteristics of high throughput, microminiaturization, integration and automation of the platform also meet the application requirements of the industrialized drug screening technology, and the platform is expected to be widely applied to drug testing.
It should be understood that the foregoing detailed description of the present invention is provided for illustration only and is not limited to the technical solutions described in the embodiments of the present invention, and those skilled in the art should understand that the present invention may be modified or substituted for the same technical effects; as long as the use requirement is met, the invention is within the protection scope of the invention.
Claims (2)
1. A centrifugal microfluidic system for high-flux drug testing comprises a chip, and is characterized in that,
the chip comprises three layers of PDMS: the three layers of PDMS are concentric circles and have the same diameter, and the three layers of PDMS are bonded, sealed and connected; the top PDMS layer is provided with gel fluid injection holes (1), microgel crosslinking reagent injection holes (2) and drug injection holes (3) which are distributed in a surrounding manner; the method comprises the steps that a surrounding type concentration gradient forming channel (4), microgel collecting liquid pond through holes (5) and gel fluid channels (6) which uniformly radiate outwards are formed in PDMS of an intermediate layer, the gel fluid channels (6) are communicated with the microgel collecting liquid pond through holes (5), concentration gradient outlets (7) of the surrounding type concentration gradient forming channel (4) are communicated with the microgel collecting liquid pond through holes (5), the number of the concentration gradient outlets (7) is the same as that of the microgel collecting liquid pond through holes (5), and drug injection holes (3) are communicated with intermediate layer holes of the intermediate layer; the bottom PDMS is a flat plate layer;
the chip is used for three-dimensional culture of two cells or three cells and high-flux drug testing; for three-dimensional culture of two cells and high-throughput drug testing, the gel fluid channel (6) comprises a plurality of closely adjacent gel channel groups distributed in groups, each channel group consisting of two closely adjacent gel channels (9); for three-dimensional culture of three cells and high-throughput drug testing, the gel fluid channel (6) comprises a plurality of closely adjacent gel channel groups distributed in groups, each channel group consisting of three closely adjacent gel channels (9);
the surrounding type concentration gradient forming channel (4) is formed by a plurality of circumferentially distributed liquid separating ports (11), liquid collecting ports (12) and a serpentine mixing channel (13) step by step, and can lead the initial two fluids to form discontinuous concentration gradient after repeated diversion, mixing and merging of the micro channels according to the laminar flow principle;
the using method of the centrifugal microfluidic system for high-flux drug testing comprises the following steps:
step 1.1: dispersing target cells in sodium alginate gel, introducing into a syringe, pushing the syringe by a microinjection pump, injecting gel fluid of the dispersed cells into the gel fluid channel (6), and gelling CaCl of sodium alginate by the microinjection pump 2 Injecting into the microgel collecting liquid pool through hole (5); the chip is fixed on a centrifugal turntable of a spin coater, when the chip rotates at a high speed, the centrifugal force applied to the gel fluid in the gel fluid channel (6) overcomes the surface tension to break from an outlet to form independent liquid drops, and the independent liquid drops and the gel cross-linking reagent CaCl in a through hole (5) of the microgel collecting liquid pool are connected with each other through the centrifugal turntable 2 Cross-linking polymerization, forming biphasic gel particles or triphasic gel particles for encapsulating cells in the microgel collecting liquid pool through holes (5);
step 1.2: caCl in a through hole (5) of the microgel collecting liquid pool 2 Changing to fresh culture medium;
step 2.1: introducing the same medicine into two medicine injection holes (3) at the same pushing speed through a microinjection pump respectively or introducing the same medicine into a high-concentration solution and a low-concentration solution respectively; the concentration gradient of the same medicine or the concentration of two combined medicines with different proportions is obtained through the surrounding type concentration gradient forming channel (4), the medicine continuously enters the microgel collecting liquid pond through hole (5) through the concentration gradient outlet (7) and acts with cells in gel particles, and at the moment, the liquid outlet of the microgel collecting liquid pond through hole (5) keeps liquid flow in the liquid pond stable;
step 2.2: and evaluating the effects of different gradient drugs on cells and the expression condition of related proteins through dead living staining and immunofluorescence experiments, and screening the medication scheme.
2. The method of using a centrifugal microfluidic system for high-throughput drug testing according to claim 1, wherein the centrifugal revolution of gel particle forming process is 500-5000 r/min for 0.5-2min.
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