A microfluidic chip for high-throughput screening of multi-drug combinations and its application

Abstract

The present invention discloses a microfluidic chip for high-throughput screening of multiple drug combinations and its application. The microfluidic chip is composed of multiple units connected in parallel, and each unit is formed by multiple cell culture chambers connected in series by multiple parallel microfluidic channels. The cell culture chambers in a single unit can all achieve independent cell culture. The multiple parallel microfluidic channels are independent of each other and are all connected to the cell culture chambers in series. By passing drugs into the microfluidic channels, a concentration gradient can be formed in the culture chambers in series, so that the drug concentration in each culture chamber is different. Since the multiple parallel microfluidic channels are relatively independent, passing different concentrations of drugs into different channels can make different drugs in the cell culture chamber have different concentrations. The chip can be used to study the effects of different drug concentration combinations on cells and thus screen the drug efficacy.

Description

Technical Field

[0001] The present invention relates to the field of microfluidics, and in particular to a microfluidics chip and a control method for high-throughput screening of drug combinations. The chip can study the effects of multiple drugs, such as three drugs mixed at different concentrations, on cells, thereby enabling high-throughput screening of drugs. Background Art

[0002] Traditional multi-drug combination screening studies rely primarily on multiwell plates. These methods utilize automated liquid transfer devices (e.g., robotic arms) or manual manipulation to transport reagents and liquids, and use microscopy to visualize cellular assays [1-3]. However, these methods are complex to operate and the automation equipment is expensive, making them unsuitable for use in small research centers or laboratories.

[0003] [1]Chen L,Ji Y,Li A,et al.High-throughput drug screening identifies fluoxetine as a potential therapeutic agent for neuroendocrine prostatecancer.Front Oncol.2023;13:1085569.Published 2023Mar 13.doi:10.3389 / fonc.2023.1085569

[0004] [2]Oudebrouckx G,Goossens J,Bormans S,Vandenryt T,Wagner P,ThoelenR.Integrating Thermal Sensors in a Microplate Format:Simultaneous Real-TimeQuantification of Cell Number and Metabolic Activity.ACS Appl MaterInterfaces.2022;14(2):2440-2451.doi:10.1021 / acsami.1c14668

[0005] [3]Dasovich M, Zhuo J, Goodman JA, et al. High-Throughput Activity Assay for Screening Inhibitors of the SARS-CoV-2Mac1 Macrodomain.ACS ChemBiol.2022;17(1):17-23.doi:10.1021 / acschembio.1c00721 Summary of the Invention

[0006] In view of the defects in the existing technology, the present invention provides a microfluidic chip for high-throughput screening of multiple drug combinations.

[0007] To this end, the microfluidic chip body provided by the present invention includes a fluid layer and a control layer;

[0008] The fluid layer is provided with an inlet unit, a plurality of drug screening units, a first main channel, a second main channel, a first outlet and a second outlet; wherein: the inlet unit includes a plurality of inlets; the drug screening unit includes a plurality of cell culture chambers, and the plurality of cell culture chambers are connected in series through a plurality of microfluidic channels, and the plurality of microfluidic channels are arranged in parallel;

[0009] The first outlet and the second outlet are arranged on one side of the chip; the first main channel and the second main channel are arranged in parallel, and one end of each of the first main channel and the second main channel is connected to the first outlet and the second outlet respectively;

[0010] The inlet unit and the plurality of drug screening units are located between the first main channel and the second main channel, and the inlet unit and the plurality of drug screening units are sequentially connected in parallel; wherein the plurality of inlets are connected to the other ends of the first main channel and the second main channel, and the plurality of inlets are connected in parallel between the first main channel and the second main channel; one end of the plurality of microfluidics in the drug screening unit is connected to the first main channel and the other end is connected to the second main channel, and the plurality of microfluidics are connected in parallel between the first main channel and the second main channel;

[0011] The control layer is provided with a plurality of control liquid inlets, a plurality of inlet control valve cavities, a plurality of cell culture chamber control valve cavities, a plurality of microfluidic channel control valve cavities and a plurality of outlet control valve cavities;

[0012] The control layer is arranged on the fluid layer, and a diaphragm is provided between the control layer and the fluid layer; at the same time, an inlet control valve cavity is provided on the passage where each inlet connects to the first main channel and the second main channel, and each inlet control valve cavity is connected to a control liquid inlet and controlled by an independent control liquid inlet; a cell culture chamber control valve cavity is provided on the serial microfluidic channel between adjacent cell culture chambers, and the cell culture chamber control valve cavity located on the same microfluidic channel is connected to the same control liquid inlet and controlled by the same control liquid inlet; a microfluidic channel control valve cavity is provided on the passage where each microfluidic channel connects to the first main channel and the second main channel, and each microfluidic channel control valve cavity is connected to a control liquid inlet; an outlet control valve cavity is provided on the passage where the two outlets connect to the first main channel and the second main channel, and the two outlet control valve cavities are connected to each other with a control liquid inlet and are controlled by an independent control liquid inlet.

[0013] An optional solution is that the number of microfluidic channels in the drug screening unit is the same as the number of types of drugs to be screened.

[0014] An optional solution is that the number of microfluidic channels in the drug screening unit is two or three.

[0015] An optional solution is that the number of inlets in the inlet unit is the same as the number of drug types to be screened.

[0016] Optionally, there are two or three inlets.

[0017] An optional solution is that the dimensions of the first main channel, the second main channel and the microfluidic channel are 100 μm in width and 25 μm in height; the dimensions of the cell culture chamber are 1500 μm in length×400 μm in width×150 μm in height.

[0018] An optional solution is that each microfluidic channel is connected to the passage between the first main channel and the second main channel, multiple structurally adjacent microfluidic channels share a microfluidic channel control valve, each microfluidic channel control cavity is connected to a control liquid inlet, and each microfluidic channel is controlled by multiple control liquid inlets.

[0019] An optional solution is that the size of each control valve cavity is 100 μm in width × 25 μm in height; the size of the microchannel between each control valve cavity and the control liquid inlet is 30 μm in width × 25 μm in height.

[0020] An optional solution is that the number of the drug screening units is 2 to 8.

[0021] The present invention also provides a method for high-throughput screening of a multi-drug combination, wherein the drug combination comprises at least drug 1 and drug 2; the method uses the above-mentioned microfluidic chip to perform high-throughput screening of the multi-drug combination; the method comprises:

[0022] (1) Substrate introduction: by controlling the corresponding inlet, outlet, microfluidic channel control valve chamber and cell culture chamber control valve chamber, each cell culture chamber is filled with a substrate for multi-drug combination screening; the substrate is a microorganism or cell;

[0023] (2) Drug introduction: by controlling the opening and closing of the corresponding inlet, outlet, control valve chamber of the corresponding drug screening unit microfluidic channel, and control valve chamber of the cell culture chamber, each single drug solution is added to the cell culture chamber through different microfluidic channels, so that multiple cell culture chambers in the same drug screening unit are filled with multiple drug solutions of different concentration combinations;

[0024] The injection method for one of the drug screening units includes:

[0025] (2.1) Controlling the opening of an inlet, a control valve cavity of a microfluidic channel A in the drug screening unit, and a cell culture chamber control valve cavity on the same microfluidic channel A; controlling the opening of the outlet control valve cavity on the first main channel; and closing the outlet control valve cavity on the second main channel; allowing a solution containing drug 1 to enter and flow through each cell culture chamber via the inlet, the second main channel, and the opened microfluidic channel; and then first closing each cell culture chamber control valve cavity on the microfluidic channel A, so that a concentration gradient of drug 1 is formed in each cell culture chamber, and the concentration of drug 1 in the cell culture chamber gradually decreases along the flow direction; and then closing the injection control valve cavity of the microfluidic channel A;

[0026] (2.2) adding a solution containing drug 2 to each cell culture chamber via another microfluidic channel B and the first main channel or another microfluidic channel B and the second main channel in the same drug screening unit using the method described in (2.1), so that a concentration gradient of drug 2 is formed in each cell culture chamber;

[0027] (3) The substrates are cultured independently using multi-drug solutions of different concentrations, and then the multi-drug combinations are screened by observing the changes in the substrates in each cell culture chamber.

[0028] The microfluidic chip of the present invention can be used for high-throughput screening of multiple drug combinations. By introducing drugs into the microfluidic channel, a concentration gradient can be formed in the cell culture chamber, so that the drug concentration in each cell culture chamber is different, thereby providing a more complex and realistic drug environment. In addition, the independent cell culture chamber design allows comparison between different drug concentrations, providing a powerful tool for studying the effects of different drug concentration combinations on cells, and thus more effectively screening and evaluating the efficacy of drugs. The innovation of this technology lies in the combination of microfluidics and drug screening needs, providing new ways and means for efficient and accurate drug development.

[0029] The microfluidic chip of the present invention is designed with a serial cell culture chamber structure, so that different cell culture chambers have different drug concentrations, which can simulate a more complex drug environment and is closer to the actual situation in vivo.

[0030] The microfluidic chip of the present invention combines microfluidic technology with drug screening needs, provides a new approach and tool for drug research and development, and helps to more effectively evaluate the efficacy and safety of drugs.

[0031] The microfluidic platform of the present invention consumes minimal sample and reagents, reduces analytical costs, and enables precise control of the volume of nanoscale slurries. In combination with MATLAB programming, it achieves a high level of system integration and automation, enabling the construction of a controllable biological microenvironment. Therefore, it is valuable to compare the proposed microfluidic platform with existing high-throughput drug screening methods to highlight the advantages and uniqueness of the proposed device. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Schematic diagram of the microfluidic chip structure of an embodiment of the present invention.

[0033] Figure 2 Schematic diagram of the structure of a microfluidic channel control system in a microfluidic chip according to an embodiment of the present invention.

[0034] Figure 3 3D schematic diagram of the microfluidic chip in an embodiment of the present invention and a COMSOL finite element analysis software simulation schematic diagram; Figure 3 (a) uses green, red, and blue to represent the diffusion of three drugs through the cell culture chamber array (the white translucent rectangle in the figure is the cell culture chamber); (b) The diffusion of three drugs in the cell culture chamber (the white translucent rectangle in the figure is the cell culture chamber); (c) is the simulated injection direction.

[0035] Figure 4 These are the test results of a simulation model with multiple cell culture chambers in series; Figure a is a numerical simulation showing the dynamic process of the drug concentration gradient being generated and reaching equilibrium after 24 hours, from top to bottom, the concentration diffusion change of the middle microfluidic channel inlet, the concentration diffusion change after 24 hours of diffusion of the middle microfluidic channel inlet, the concentration diffusion change of the lower microfluidic channel inlet, and the concentration diffusion change after 24 hours of diffusion of the lower microfluidic channel inlet; the color change in the figure reflects the concentration change, and the specific relationship can be seen in the coclebar of Figure a; Figure b is the estimated concentration of drug molecules at different positions and different time points in the cell culture chamber, where the horizontal axis is the 5 culture chambers in the same column, and the vertical axis is the expected concentration change; the legend indicates the middle microfluidic channel injection (middle) and the lower microfluidic channel injection (side) as well as the time of injection.

[0036] Figure 5Figure 1 shows the effects of multiple growth factors and neural stem cells studied and analyzed using the chip of the present invention in the examples; the horizontal axis in the figure represents the different drug components and concentrations in the combination drug, and the vertical axis represents the differentiation rate of neural stem cells after the combination drug is applied; the four figures from left to right are: the differentiation rate of NSCs cells with only culture medium added to the cavity (control); the differentiation rate of NSCs cells under the stimulation of the combination drug with EGF as the main drug in the cavity; the differentiation rate of NSCs cells under the stimulation of the combination drug with FGF as the main drug in the cavity; and the differentiation rate of NSCs cells under the stimulation of the combination drug with PDGF as the main drug in the cavity. DETAILED DESCRIPTION

[0037] Unless otherwise specified, the scientific and technical terms used herein are understood according to the knowledge of ordinary technicians in the relevant fields.

[0038] The inventors discovered that when a liquid containing a certain concentration of drug flows sequentially through multiple cell culture chambers connected in series by microfluidic channels, a concentration gradient forms within the cell culture chamber under certain conditions (including reasonable flow rate, injection time, channel width, and cell culture chamber size), and the drug concentration within the cell culture chamber decreases along the direction of the flow. The specific research plan uses COMSOL finite element analysis software for simulation, and the specific simulation method is as follows:

[0039] The actual structure of the simulation chip consists of three microfluidic channels connected in sequence to five cell culture chambers. The dimensions of the microfluidic channels are 100 μm wide and 25 μm high. The dimensions of the cell culture chamber are 1500 μm long, 400 μm wide, and 150 μm high. A set of two-dimensional graphics were drawn in the software. The selected physical fields were laminar flow field (SPF) and dilute substance transport field (TDS), with the initial concentration of the dilute substance transport field being 1 mol / m^3. During the laminar flow simulation, only one column corresponding to the inlet and outlet was introduced at a time. Normal inflow velocities of 3 mm / s, 5 mm / s, and 10 mm / s were simulated at the inlet. Different inflow times (different liquid replacement rates) were simulated for different inflow velocities. For example, when the inflow velocity was 5 mm / s, the inflow stopped after 3.5 s, 4 s, and 4.5 s, respectively. At this time, the diluted substances already in the chamber could diffuse freely. The simulation results were compared and analyzed, as shown in Figure 4, where (a) the numerical simulation shows the dynamic process of drug concentration gradient generation and reaching equilibrium after 24 hours; (b) the estimated concentration of drug molecules at different locations and time points in the culture chamber.

[0040] Based on the above research findings, the present invention adopts a reasonable cell culture chamber structure and pathway control concept to form a stable concentration difference or concentration gradient in different cell culture chambers.

[0041] Based on the microfluidic chip structure of the present invention, during sample injection, the flow rate of liquid is controlled by controlling the flow rate of liquid through the serial culture chambers through inlet and outlet control valves, thereby forming a concentration gradient within the cell culture chambers. In addition, by placing control valves between the serial cell culture chambers, the drug concentration within the cell culture chambers can be stabilized after the concentration gradient is formed.

[0042] In order to ensure the concentration gradient of multiple drugs and prevent the later-input drug 2 from destroying the concentration state of the already-input drug 1 in the cavity, the chip of the present invention adopts multiple parallel microfluidic channels. After drug 1 is injected through a microfluidic channel, the entrance and exit of the microfluidic channel and the connection between the channel and adjacent cell cultures need to be closed to ensure the stability of the concentration of drug 1 in the cell culture cavity; then, drug 2 is injected through another microfluidic channel. In this way, by injecting different drugs through different microfluidic channels, it is theoretically possible to form a situation where multiple drugs are mixed in multiple groups of concentration gradients.

[0043] It should be noted that in the specific scheme, the cell culture chamber size, microfluidic channel inner diameter, number of microfluidic channels, structural spacing between adjacent channels, and injection volume in each drug screening unit must be reasonably set to ensure that when the drug is injected into the current microfluidic channel, it will not affect the concentration of the drug that has entered the cell culture chamber through other microfluidic channels. This ensures that the drug injected into each parallel microfluidic channel can be evenly diffused and distributed in the cell culture chamber. In the specific scheme, the reasonable setting of these parameters can be obtained through simulation in relevant software.

[0044] When using the chip of the present invention with multiple microfluidic channels connected in series with multiple cell culture chambers to conduct different drug mixing studies, in order to achieve the combination of multiple drugs and multiple concentration values, the chip of the present invention is designed with two main channels and multiple drug screening units, and the number of microfluidic channels in each drug screening unit is the same as the number of drug types. Taking a drug screening unit with three microfluidic channels in series with multiple cell culture chambers and eight drug screening units as an example, when sampling, the liquid flow directions in the eight units are drug screening unit 1 (↓↓↓), drug screening unit 2 (↓↓↑), drug screening unit 3 (↓↑↑), drug screening unit 4 (↓↑↓), drug screening unit 5 (↑↓↓), drug screening unit 6 (↑↓↑), drug screening unit 7 (↑↑↑), and drug screening unit 8 (↑↑↓) (↓ represents that the upper main channel of one of the microfluidic channels is closed and the lower main channel is opened, and the sample flows from the upper part of the microfluidic channel through the cell culture chambers of the corresponding unit, thereby forming a drug concentration decrease from top to bottom; ↑ represents that the upper main channel is opened and the lower main channel is closed, and the sample flows from the lower part of the microfluidic channel through the cell culture chambers of the corresponding unit, thereby forming a drug concentration increase from top to bottom). As the drug concentration in the serial cell culture chambers gradually decreases with the direction of fluid movement, different combinations of multi-drug concentration gradients are achieved in each drug screening unit.

[0045] When implementing the above control method, the liquids flowing into each microfluidic channel or the ways in which the liquids flow in are different, so the connection between each microfluidic channel and the main channel and the switches between adjacent cell culture chambers need to be independently controlled.

[0046] In a specific solution, if a separate control valve is set for each channel, too many valves will be operated, which is not conducive to the overall design and control of the chip. To solve this problem, in some solutions, the control valve chambers between adjacent cell culture chambers on the same microfluidic channel are controlled by the same control liquid inlet. In addition, multiple sets of independently controllable valve systems are used to control the switching of the corresponding control valve chambers between each microfluidic channel and the main channel. The principle is "multiple controls one", such as "three controls one". That is, multiple control valve chambers are set on each microfluidic channel, and the microfluidic channel will only be connected when multiple valve chambers are open. That is, each microfluidic channel is connected to the first main channel and the second main channel. Multiple structurally adjacent microfluidic channels share a microfluidic channel control valve, and each microfluidic channel control chamber is connected to a control liquid inlet. Each microfluidic channel is controlled by multiple control liquid inlets.

[0047] The high-throughput screening described in the present invention refers to the design of multiple parallel drug screening units, each containing multiple cell culture chambers and microfluidic channels, and the realization of high-throughput screening of multiple drug combinations through multi-concentration gradient control and independent culture, which greatly improves the screening efficiency. The concentration gradient control refers to the formation of a concentration gradient in the serial cell culture chambers by introducing a single drug solution into a single microfluidic channel, so that the drug concentration in each cell culture chamber is different, thereby realizing single and multiple drug concentration gradient control. The independent culture means that the cell culture chambers in each drug culture unit are independent culture environments, ensuring the independence of experiments under different drug concentration conditions.

[0048] Closing the control valve cavity in the present invention involves adding control liquid through the corresponding control liquid inlet, increasing the pressure within the control valve cavity and squeezing the diaphragm (the diaphragm is typically made of PDMS, which deforms upon compression and pushes the extrusion channel upward to form valve control), thereby closing the passage below the diaphragm. Opening the control valve cavity involves withdrawing the control liquid from the corresponding control valve cavity to reduce the pressure, allowing the diaphragm to recover and open the corresponding passage. Water is often used as the control liquid.

[0049] Example 1:

[0050] See also Figure 1 and 2As shown, the fluid layer of the microfluidic chip of this embodiment is provided with three inlets (r1, r2, r3), two main channels (D1, D2), two outlets (r7, r8), three inlets (r1, r2, r3), and eight parallel drug screening units S. Each drug screening unit is provided with three microfluidic channels in parallel and five cell culture chambers (Q1, Q2, Q3; only three are shown in the figure) connected in series through the three microfluidic channels; two outlets are arranged in parallel on one side of the chip; two main channels are arranged in parallel, and one end of each main channel is connected to the two outlets respectively;

[0051] An inlet unit with three inlets and eight drug screening units are located between the two main channels. The three inlets are connected to the other ends of the first and second main channels, and multiple inlets are connected in parallel between the two main channels. Multiple microchannels are connected at both ends to the two main channels, and multiple microchannels are connected in parallel between the two main channels.

[0052] The fluid channels (main channel and microchannel) of the fluid layer of this embodiment are: width 100 μm × height 25 μm, and the dimensions of the cell culture chamber are: length 1500 μm × width 400 μm × height 150 μm;

[0053] The chip control layer is provided with multiple control valve chambers (f1, f2, f3, f4, f5, f6), multiple control liquid inlets (r4, r5, r6), multiple cell culture chamber control valve chambers (f7, f8, f9, f10, f11, f12), multiple microfluidic channel control valve chambers and multiple outlet control valve chambers (f13, f14); the dimensions of the microchannels connecting the control liquid inlets and the valve chambers in the control layer are: 30 μm wide × 25 μm high, and the dimensions of the valve chambers are: 100 μm wide × 25 μm high.

[0054] The chip control layer control valve is located below the fluid channel and a diaphragm is provided between them. Each inlet is connected to the upper and lower main channels and is provided with (f1, f2, f3, f4, f5, f6). The cell culture chamber control valve chamber (f7, f8, f9, f10, f11, f12) is provided between adjacent cell culture chambers on the same microfluidic channel.

[0055] Each control valve cavity is connected to the corresponding control fluid inlet via the control layer microchannels. During operation, the control layer microchannels are filled with water. External solenoid valves control the gas pressure, which increases the liquid pressure within the control layer microchannels. This causes the diaphragm between the control valve and the fluid channel of the upper fluid layer to deform spatially, thereby controlling the opening and closing of the fluid layer microchannels.

[0056] The 24 microfluidic channels of this embodiment need to be controlled independently, such as Figure 2As shown, the chip is equipped with seven groups of "three-control-one" valve control systems (①-⑦) that can be independently controlled by separate control liquid inlets at the connection points of all microfluidic channels (F1, F2), specifically including: 1-fifteen-valve chamber (fifteen-valve chamber represents the microfluidic channel control valve chamber corresponding to 15 microfluidic channels), 2-fourteen-valve chamber, 3-five-valve chamber, 4-first four-valve chamber, 5-second four-valve chamber, 6-first single valve chamber, 7-third four-valve chamber, 8-first triple valve chamber, 9-second single valve chamber, 10-third single valve chamber, 11- The fourth single valve chamber, 12-second triple valve chamber, 13-fifth single valve chamber, 14-sixth single valve chamber, 15-seventh single valve, 16-eighth single valve, 17-ninth single valve, 18-tenth single valve, 19-eleventh single valve, 20-twelfth single valve, 21-thirteenth single valve chamber, 22-fourteenth single valve chamber, 23-fifteenth single valve chamber, 24-first double valve chamber, 25-sixteenth single valve chamber, and 26-second double valve chamber are all examples of this valve control system. Under the control of this channel, only when all three valves are open can the channel connect to the main channel. By rationally distributing the control valves in series and distributing the valve control system in permutations and combinations, a seven-way control system can achieve on-off control of the 24 independent microfluidic channels in the eight drug screening units K1-8. For example, by opening control valve chambers 26, 20, and 1 corresponding to the control fluid inlets of ①, ②, and ⑦, and closing the remaining valve chambers at the control fluid inlets of the remaining groups, the leftmost microfluidic channel can be opened.

[0057] The method for high-throughput screening of multiple drug combinations using the above-mentioned microfluidic chip comprises the following steps:

[0058] Step 1: inject liquid into the chip control layer channel to ensure that the opening and closing of the fluid layer control valve can be controlled by the control pressure generated in the control layer microchannel.

[0059] Step 2: Open all control valve chambers in the chip.

[0060] Step three: inject cell suspension from the inlet of the chip, so that the cell suspension flows into the main channel above the chip and into the cell culture chambers of all drug screening units. After the cell culture chamber is filled with cell suspension, close all control valve chambers to ensure that the cells in the cell culture chamber are stable and not disturbed by the flow field.

[0061] Step 4: Execute steps 2-3, inject cell-free culture medium from the inlet of the chip, control the culture medium to slowly flow into each cell culture chamber of the chip, and then close all control valve chambers to allow the cells in the cell culture chamber to adhere to the wall in a stable environment.

[0062] Step 5: Inject drugs into the cell culture chamber, insert syringes filled with different drug solutions at the three inlets for sampling; this step can control the drug to flow from the upper main channel into the drug screening unit and out of the lower main channel (the drug concentration in the upper cell culture chamber is greater than the drug concentration in the lower cell culture chamber), or it can control the drug to flow from the lower main channel into the drug screening unit and out of the upper main channel (the drug concentration in the lower cell culture chamber is greater than the drug concentration in the upper cell culture chamber). The following steps are used from top to bottom as an example to illustrate the operation method. All drug screening units in the chip have the same liquid inlet method. The following steps take the first drug screening unit as an example:

[0063] 5.1. Open the control valve chamber above inlet r2, open all control valve chambers of the left microfluidic channel d4, open the control valve chamber of outlet r8, and close the remaining control valve chambers; allow drug 1 to flow from top to bottom through all cell culture chambers of drug screening unit K1 through inlet r2, the upper main channel, and microfluidic channel d4.

[0064] 5.2. Close all control valve chambers of the microfluidic channel on the left side of the drug screening unit K1 to stabilize the concentration difference of drug 1 in each cell culture chamber.

[0065] 5.3. Open the control valve chamber above the inlet r1, open the control valve chamber at the outlet r7, flush the remaining medicine 1 in the upper main channel, and close the control valve chamber at the outlet r7 after flushing.

[0066] 5.4. Open the control valve chamber above the inlet r7, open all the internal control valve chambers of the middle microfluidic channel d5 of the drug screening unit K1, open the control valve chamber of the chip outlet r8, and close the remaining control valve chambers to allow drug 2 to flow from top to bottom through all cell culture chambers of the drug screening unit K1 through the inlet r1, the upper main channel, and the microfluidic channel d5.

[0067] 5.5. Close all control valves in the middle microfluidic channel of the drug screening unit to stabilize the concentration difference of drug 2 in different culture chambers.

[0068] The above steps are for the drug to pass through the serial cell culture chamber from top to bottom. For example, if drug 3 is injected from bottom to top, the following steps are used:

[0069] 5.6. Open the control valve cavity below the inlet r3 and inject the drug 3. Open the control valve cavity of the main channel outlet r8 below the chip to flush the remaining drug 2 in the pipeline. After flushing, close the upper main channel outlet control valve.

[0070] 5.7. Open the control valve chamber below inlet r3, open all internal control valves of the microfluidic channel d6 on the right side of the drug screening unit, and open the control valve chamber at the main channel outlet above the chip to allow drug 3 to flow from top to bottom through all cell culture chambers.

[0071] 5.8. Close all control valve chambers of the microfluidic channel on the right side of the drug screening unit to stabilize the concentration difference of drug 3 in different culture chambers so that the concentration of drug 3 in a single culture chamber does not change.

[0072] 5.9. After the intracellular drug diffusion stabilizes, use a fully automated microscope to image the cells in the chip cell culture chamber, and analyze the effects of the drug on the cells through the cell images.

[0073] The chip structure of this embodiment is simulated in COMSOL finite element analysis software. Figure 3 ,

[0074] like Figure 3 As shown in a, multiple culture chamber arrays are controlled separately by valve control, wherein the culture chambers are connected by three microfluidic channels; the formation of drug gradient is due to the drug flowing along the path shown ( Figure 3 c) enters the culture chamber array in the form of displacement. As the drug entry time increases, the longer the displacement time along the top chamber of the path, the higher the concentration; the displacement time of each chamber along the path gradually decreases, resulting in concentration differences and ultimately forming a drug gradient. Therefore, by specifying the drug injection time and input flow rate, a similar drug gradient can be formed along the path. In addition, Figure 3 After the simulation injection as shown in c, Figure 3 As shown in Figure b, the liquid displacements in the three microfluidic channels have almost no mutual influence. The microfluidic channels are individually controlled by valves with a response speed of less than 0.1 second, which can achieve precise drug delivery time and ensure the accuracy and repeatability of each drug concentration gradient.

[0075] Example 2:

[0076] This example uses the microfluidics chip described in Example 1 to analyze the effects of multiple growth factors (epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and fibroblast growth factor (FGF)) at different concentration combinations on neural stem cells (NSCs) (neural stem cells isolated and cultured on day 16 from the embryonic forebrain of SD (Sprague Dawley) rats and Hes5-GFP / Dcx-RFP double transgenic mice).

[0077] Among them, epidermal growth factor (EGF) promotes the proliferation and differentiation of neural stem cells. Platelet-derived growth factor also plays an important regulatory role in the nervous system, promoting the migration and proliferation of neural stem cells and participating in the generation and development of neurons and glial cells. Fibroblast growth factor can stimulate the proliferation and differentiation of neural stem cells, promoting the generation of neurons and the development of the nervous system. In addition, it can also promote the migration of nerve cells and the formation of synapses.

[0078] The solvents for each drug sample solution are: culture medium (KnockOut TM DMEM / F-12CTS TM Catalog number: A1370801); concentrations were: 1 μg / ml PDGF; 0.2 μg / ml FGF; 500 ng / ml EGF; each injection lasted 4 s, with a flow rate of 5 mm / s; each injection volume was approximately 0.05 ml. After a single injection, the control valve between adjacent cell culture chambers was closed before the next injection.

[0079] The sample was injected using the method described in Example 1. After stabilization, the drug concentration in each culture chamber was represented by EGF-1, -2, -3, -4, and -5, and EGF-1, -2, -3, -4, and -5 represented the EGF concentration from high to low. FGF and PDGF were represented in the same way. Each growth factor was introduced into the drug screening unit of the chip through three inlets. The number of drugs introduced was different. One of the drug screening units was a blank control group. The sample was only injected with the culture medium (KnockOut TM DMEM / F-12CTS TM Catalog number: A1370801). As the fluid was directed through the culture chamber array, a concentration gradient was generated. The estimated concentration gradients for EGF-1, -2, -3, -4, and -5 were approximately 110 ng / ml, 100 ng / ml, 85 ng / ml, 70 ng / ml, and 55 ng / ml; for FGF-1, -2, -3, -4, and -5, they were 44 ng / ml, 40 ng / ml, 34 ng / ml, 28 ng / ml, and 22 ng / ml, respectively; and for PDGF-1, -2, -3, -4, and -5, they were approximately 220 ng / ml, 200 ng / ml, 170 ng / ml, 140 ng / ml, and 110 ng / ml, respectively. After the experimental group sample injections were completed, the control valves between adjacent cell culture chambers were closed, and the microfluidic chip was placed on a microscope equipped with a culture system and observed continuously for at least 48 hours. Data were then collected and recorded.

[0080] Combine Figure 5As shown, the behavior of NSCs maintained on the chip for 48 hours under different drug conditions revealed that: (1) the negative effects of excessive use of a single growth factor (i.e., differentiation) can be mediated by excessive use of another growth factor; (2) although low-concentration addition of a single growth factor helps maintain the stemness of NSCs, low-concentration addition of growth factors has no significant effect on control and differentiated NSCs, except for PDGF-5; (3) NSCs often respond sluggishly under complex environmental conditions, i.e., under the action of multiple growth factors. Notably, NSC differentiation induced by excessive EGF and FGF can be mediated by low concentrations of PDGF. However, NSC differentiation induced by excessive PDGF is not interfered with by low doses of EGF and FGF. These results suggest that PDGF has a stronger effect as a promoter of NSC stemness or differentiation than other growth factors. We suspect that the signaling pathway activated by PDGF is digital, in which the cascade reaction is triggered by a small amount of stimulation. The signaling pathways of other growth factors are similar, and their amplitude is concentration-dependent.

Claims

1. A microfluidic chip for high-throughput screening of multiple drug combinations, characterized in that: The microfluidic chip body includes a fluid layer and a control layer; The fluid layer is provided with an inlet unit, a plurality of drug screening units (S), a first main channel (D1), a second main channel (D2), a first outlet (r7) and a second outlet (r8); wherein: the inlet unit includes a plurality of inlets (r1, r2, r3); the drug screening unit includes a plurality of cell culture chambers (Q1, Q2, Q3), and the plurality of cell culture chambers are connected in series through a plurality of microfluidic channels (d4, d5, d6), and the plurality of microfluidic channels are arranged in parallel; The first outlet and the second outlet are arranged on one side of the chip; the first main channel and the second main channel are arranged in parallel, and one end of each of the first main channel and the second main channel is connected to the first outlet and the second outlet respectively; The inlet unit and the plurality of drug screening units are located between the first main channel and the second main channel, and the inlet unit and the plurality of drug screening units are sequentially connected in parallel; wherein the plurality of inlets are all connected to the other ends of the first main channel and the second main channel, and the plurality of inlets are connected in parallel between the first main channel and the second main channel; the plurality of microfluidic channels in the drug screening unit are connected to the first main channel at one end and to the second main channel at the other end, and the plurality of microfluidic channels are connected in parallel between the first main channel and the second main channel; The control layer is provided with a plurality of control liquid inlets, a plurality of inlet control valve cavities, a plurality of cell culture chamber control valve cavities, a plurality of microfluidic channel control valve cavities and a plurality of outlet control valve cavities; The control layer is arranged on the fluid layer, and a diaphragm is arranged between the control layer and the fluid layer; at the same time, each inlet is connected to the first main channel and the second main channel. The passage (d1, d2, d3) is provided with an inlet control valve cavity (f1, f2, f3, f4, f5, f6), and each inlet control valve cavity is connected to a control liquid inlet and is controlled by an independent control liquid inlet; the series microfluidic channel between adjacent cell culture chambers is provided with a cell culture chamber control valve cavity (f7, f8, f9, f10, f11, f12), and is located in the same microfluidic channel. The cell culture chamber control valve cavity on the flow channel is connected to the same control liquid inlet (r4, r5, r6) and is controlled by the same control liquid inlet; a microfluidic channel control valve cavity is provided on the passage connecting each microfluidic channel to the first main channel and the second main channel, and each microfluidic channel control valve cavity is connected to the control liquid inlet; an outlet control valve cavity (f13, f14) is provided on the passage connecting the two outlets to the first main channel and the second main channel, and the two outlet control valve cavities are respectively connected to the control liquid inlet and are controlled by an independent control liquid inlet.

2. The microfluidic chip for high-throughput screening of multiple drug combinations according to claim 1, characterized in that: The number of microfluidic channels in the drug screening unit is the same as the number of types of drugs to be screened.

3. The microfluidic chip for high-throughput screening of multiple drug combinations according to claim 1, characterized in that: The number of microfluidic channels in the drug screening unit is two or three.

4. The microfluidic chip for high-throughput screening of multiple drug combinations according to claim 1, characterized in that: The number of inlets in the inlet unit is the same as the number of drug types to be screened.

5. The microfluidic chip for high-throughput screening of multiple drug combinations according to claim 1, characterized in that: There are two or three inlets.

6. The microfluidic chip for high-throughput screening of multiple drug combinations according to claim 1, characterized in that: The dimensions of the first main channel, the second main channel and the microfluidic channel are 100 μm in width and 25 μm in height; the dimensions of the cell culture chamber are 1500 μm in length×400 μm in width×150 μm in height.

7. The microfluidic chip for high-throughput screening of multiple drug combinations according to claim 1, characterized in that: The dimensions of each control valve cavity are 100 μm in width and 25 μm in height; the dimensions of the microchannel between each control valve cavity and the control liquid inlet are 30 μm in width and 25 μm in height.

8. The microfluidic chip for high-throughput screening of multiple drug combinations according to claim 1, characterized in that: The number of the drug screening units is 2 to 8.

9. A high-throughput screening method for a multi-drug combination, wherein the multi-drug combination comprises at least drug 1 and drug 2; characterized in that: The method uses the microfluidic chip of claim 1 to perform high-throughput screening of a multi-drug combination; the method comprises: (1) Substrate feeding: by controlling the corresponding inlet, outlet, microfluidic channel control valve chamber and cell culture chamber control valve chamber, each cell culture chamber is filled with a substrate for multi-drug combination screening; the substrate is a microorganism or cell; (2) Drug introduction: by controlling the opening and closing of the corresponding inlet, outlet, control valve chamber of the corresponding drug screening unit microfluidic channel and the control valve chamber of the cell culture chamber, each single drug solution is added to the cell culture chamber through different microfluidic channels, so that multiple cell culture chambers in the same drug screening unit are filled with multiple drug solutions of different concentration combinations; The injection method for one of the drug screening units includes: (2.1) Controlling the opening of an inlet, a control valve chamber of a microfluidic channel in the drug screening unit, and a cell culture chamber control valve chamber on the same microfluidic channel, controlling the opening of the outlet control valve chamber on the first main channel and the closing of the outlet control valve chamber on the second main channel, allowing a solution containing drug 1 to enter and flow through each cell culture chamber via an inlet, the second main channel, and the opened microfluidic channel, and then first closing each cell culture chamber control valve chamber on the microfluidic channel, so that a concentration gradient of drug 1 is formed in each cell culture chamber, and the concentration of drug 1 in the cell culture chamber gradually decreases along the flow direction; then closing the injection control valve chamber of the microfluidic channel; (2.2) adding a solution containing drug 2 to each cell culture chamber via another microfluidic channel and the first main channel or another microfluidic channel and the second main channel in the same drug screening unit using the method described in (2.1), so that a concentration gradient of drug 2 is formed in each cell culture chamber; (3) The substrates are cultured independently using multi-drug solutions of different concentrations, and then the multi-drug combinations are screened by observing the changes in the substrates in each cell culture chamber.

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