High-throughput microfluidic chip for 3D cell automated culture and multidimensional drug efficacy evaluation and preparation method thereof
By designing a high-throughput microfluidic chip for automated 3D cell culture and multi-dimensional drug efficacy evaluation, the problems of cumbersome 3D cell culture operation and inaccurate drug screening in existing technologies have been solved, realizing efficient multi-organ drug action simulation and drug screening.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- ZHEJIANG UNIV BINJIANG RES INST
- Filing Date
- 2022-06-16
- Publication Date
- 2026-06-02
AI Technical Summary
Existing microfluidic chips cannot accurately reflect the drug effects of three-dimensional cells in drug screening, and existing three-dimensional cell culture methods are cumbersome to operate and cannot efficiently simulate the effects of drugs on multiple organs.
Design a high-throughput microfluidic chip for automated 3D cell culture and multi-dimensional drug efficacy evaluation, including a PDMS microvalve control layer, a PDMS thin film flow path chamber layer, and a PDMS microgroove layer. Through microvalve control, high-throughput culture and evaluation of cells and drugs can be achieved. It can construct various 3D cell spheroid models and perform tests on various drugs or drug concentration gradients.
It enables high-throughput and simple three-dimensional cell culture and multi-dimensional drug evaluation, and can simulate drug effects in multiple organs, thus improving the accuracy and efficiency of drug screening.
Smart Images
Figure CN115386490B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to a high-throughput 3D cell preparation and drug efficacy evaluation method, and more particularly to a high-throughput microfluidic chip for automated 3D cell culture and multi-dimensional drug efficacy evaluation and its preparation method. Background Technology
[0002] Chemotherapy is currently the most commonly used method for cancer treatment, but it often causes many side effects for patients. Therefore, early preclinical evaluation of the efficacy and side effects of chemotherapy drugs is of great significance for drug development. In the drug development cycle, preclinical drug evaluation mainly includes cell experiments and animal experiments, with cell experiments being simpler and more cost-effective than animal experiments.
[0003] Microfluidic chips offer several advantages, including low reagent consumption, high throughput, and low sample requirements, making them ideal for high-throughput drug evaluation when combined with cell culture. Published literature and technologies have already demonstrated the use of microfluidic chips for high-throughput drug evaluation of 2D cells. For example, Chinese patent CN101629143B discloses a microfluidic cell array chip for high-throughput drug screening, its fabrication method, and its applications. The chip, from top to bottom, consists of a valve-controlled channel layer, a fluid channel layer, and a glass layer suitable for cell adhesion and growth. This chip can be used to simultaneously treat multiple cell types with different drug concentrations for high-throughput cell-drug screening. The chip employs a Su-8 negative photoresist process with multiple exposures and a single development, along with multilayer PDMS bonding, to create a multilayer structure with a high aspect ratio. The microfluidic cell array chip provided by this invention enables low-reagent-consumption, high-throughput co-culture of multiple cell types, parallel analysis of the stimulatory effects of different drug concentrations on different cells, and real-time observation and detection on the chip. Specifically, microvalve technology is used to achieve drug screening at different drug concentration gradients through co-culture of multiple 2D cells. However, 2D cells lack a three-dimensional microenvironment and cannot accurately reflect the effects of drugs on in vivo cells. In some drug development processes using 2D cells, there are cases where the drug shows good efficacy in cell experiments, but poor efficacy in animal experiments.
[0004] To address the significant differences between two-dimensional cells and in vivo cells, three-dimensional cell culture has been employed in recent years for drug screening to reduce these discrepancies. Common methods for culturing three-dimensional cells include multi-well plate culture, hanging drop culture, and centrifugation. These methods are cumbersome, inefficient, and only suitable for testing single-species three-dimensional cell spheres in in vitro cell-based drug evaluation experiments, failing to simulate the drug's effects across multiple organs during actual administration. Therefore, the field of drug screening urgently needs a device and method that leverages the advantages of microfluidic chips to easily and efficiently prepare three-dimensional cells and evaluate the efficacy of drug delivery across multiple organs. Summary of the Invention
[0005] The purpose of this invention is to address the shortcomings of existing technologies by providing a high-throughput microfluidic chip for automated 3D cell culture and multi-dimensional drug efficacy evaluation, as well as its preparation method. This high-throughput drug evaluation microfluidic chip can be freely combined and used to construct multiple in vitro drug evaluation models composed of cell lines or organoids in batches, and can simultaneously perform drug evaluation tests on multiple drugs or different drug concentration gradients.
[0006] To achieve the above objectives, the technical solution adopted by the present invention to solve its technical problems is as follows:
[0007] A high-throughput microfluidic chip for automated 3D cell culture and multi-dimensional drug efficacy evaluation comprises, from top to bottom, a PDMS microvalve control layer, a PDMS thin film flow path chamber layer, and a PDMS microgroove layer;
[0008] The PDMS microvalve control layer is equipped with multiple lateral pressure valves and multiple longitudinal pressure valves; the lateral pressure valves and longitudinal pressure valves are arranged in a cross pattern; the lateral pressure valves and longitudinal pressure valves are controlled to open and close by outputting pressure to the lower membrane flow path chamber layer.
[0009] The PDMS film flow path chamber layer is provided with multiple transverse flow paths and multiple longitudinal flow paths. The two ends of the transverse flow paths are connected to the drug inlet and the drug outlet, respectively, and the two ends of the longitudinal flow paths are connected to the cell inlet and the cell outlet, respectively. The intersection of the transverse flow paths and the longitudinal flow paths forms a culture chamber.
[0010] The PDMS microgroove layer is provided with microgrooves that correspond one-to-one with the culture chambers.
[0011] Preferably, the inlet of the transverse flow path is connected to the drug inlet via a liquid inlet defoaming microcolumn and a drug inlet defoaming chamber, and the inlet of the longitudinal flow path is connected to the cell inlet via a cell homogenization microcolumn.
[0012] Preferably, there are 4 transverse pressure valves and 5 longitudinal pressure valves. The pressure inlets of the transverse and longitudinal pressure valves are located on the upper surface of the PDMS micro-valve control layer. Each transverse pressure valve consists of 5 transverse micro-valves and each longitudinal pressure valve consists of 4 longitudinal micro-valves. The transverse micro-valve has a length of 1-4 mm and a width of 1-2 mm, and the longitudinal micro-valve has a length of 1-5 mm and a width of 1-2 mm.
[0013] Preferably, the PDMS microvalve control layer is controlled by input air pressure or hydraulic pressure.
[0014] Preferably, the inner diameter of the drug inlet is 0.7-2 mm, the inner diameter of the drug outlet is 0.7-2 mm, the inner diameter of the cell inlet is 0.7-2 mm, the inner diameter of the cell outlet is 0.7-2 mm, the inner diameter of the defoaming microcolumn is 0.05-0.3 mm, the inner diameter of the cell homogenizing microcolumn is 0.1-0.3 mm, the dimensions of the culture chamber are 2-3 mm in length and 2-3 mm in width, the width of the transverse flow path is 0.1-1 mm, and the width of the longitudinal flow path is 0.1-1 mm.
[0015] Preferably, each microgroove array includes 19 microgrooves, with a diameter of 0.15-0.5 mm and a depth of 200-600 mm.
[0016] A method for preparing a high-throughput microfluidic chip for automated 3D cell culture and multi-dimensional drug efficacy evaluation, characterized by comprising the following steps:
[0017] Fabrication of the PDMS microvalve control layer: A silicon wafer is pretreated and cleaned with oxygen plasma. A layer of negative photoresist is then spun onto the cleaned silicon wafer. The photoresist-coated silicon wafer is then vacuum-treated to remove air bubbles from the photoresist. The bubble-removed sample is placed on a hot plate for pre-baking. The pre-baked sample is placed in the sample tray of a UV exposure machine. A mask containing the microstructure pattern of the microvalve control layer is mounted on a mask holder. The relative position of the sample and the mask is adjusted, and the sample is raised to contact the mask before UV exposure. The UV-exposed sample is placed on a hot plate for post-baking. The post-baked sample is immersed in a developer until the microstructure of the microvalve appears, then the sample is cleaned. A PDMS mixed solution is poured into the prepared silicon wafer mold, heated and cured, then peeled off from the mold, cut, and perforated at each control input port to obtain the PDMS microvalve control layer.
[0018] Preparation of PDMS thin film flow channel chamber layer: A silicon wafer is pretreated and cleaned with oxygen plasma. Positive photoresist is then spun onto the cleaned silicon wafer. The photoresist-coated silicon wafer is then vacuum-treated to remove air bubbles within the photoresist layer. The bubble-removed sample is placed on a hot plate for pre-baking. The pre-baked sample is placed in the sample tray of a UV exposure machine. A mask containing the microstructure pattern of the flow channel chamber layer is mounted on a mask holder. The relative position of the sample and the mask is adjusted, and the sample is raised to contact the mask before UV exposure. The UV-exposed sample is placed on a hot plate for post-baking. The post-baked sample is immersed in a developer until the microstructure of the flow channel chamber appears, then the sample is cleaned. The sample is placed on a hot plate at a suitable temperature to soften the microstructure. A PDMS mixed solution is spun onto the prepared silicon wafer sample and cured by heating to obtain the PDMS thin film flow channel chamber layer.
[0019] PDMS microgroove layer preparation: Silicon wafers are pretreated and cleaned using oxygen plasma. Negative photoresist is then spun onto the cleaned wafer. The photoresist-coated wafer is then vacuum-treated to remove air bubbles from the photoresist. The bubble-removed sample is placed on a hot plate for pre-baking. The pre-baked sample is placed in a UV exposure tray. A mask containing the microgroove layer microstructure pattern is mounted on a mask holder. The relative position of the sample and mask is adjusted, and the sample is raised to contact the mask before UV exposure. The UV-exposed sample is placed on a hot plate for post-baking. The post-baked sample is immersed in a developer until the microstructure of the microgrooves appears, then the sample is cleaned. A PDMS mixed solution is poured into the silicon wafer mold prepared in the above steps, heated to cure, and then peeled off from the mold to obtain the PDMS microgroove layer.
[0020] A high-throughput drug evaluation microfluidic chip is constructed by bonding multiple layers of PDMS: The PDMS microvalve control layer and the PDMS thin film flow path chamber layer still on the mold are simultaneously subjected to oxygen plasma treatment; the PDMS microvalve control layer and the PDMS thin film flow path chamber layer still on the mold are aligned and bonded, and then the two sample layers are peeled off from the mold and the inlet and outlet of the flow path chamber layer are perforated; the bonded sample and the PDMS microgroove layer are simultaneously subjected to oxygen plasma treatment, and then aligned and bonded.
[0021] Preferably, a steel needle is inserted into the flow path inlet / outlet and the micro-valve control source inlet and connected to a flexible tube.
[0022] Compared with the prior art, this application has at least the following obvious advantages and effects:
[0023] The high-throughput drug evaluation microfluidic chip of this invention can isolate each chamber through internal microvalves, and simultaneously change the fluid path into two different modes: cell sample introduction and drug infusion. In sample introduction mode, the high-throughput microgroove array at the bottom of the culture chamber allows different cells injected according to different flow paths to deposit and aggregate into 3D cell spheres or organoids, thereby constructing multiple 3D cell sphere models for high-throughput drug evaluation of oral drugs in vitro; in infusion mode, different drugs or different drug concentrations can be injected simultaneously, efficiently completing multi-dimensional drug screening. Attached Figure Description
[0024] Figure 1 This is a schematic diagram of the overall appearance of the high-throughput drug evaluation microfluidic chip in this invention;
[0025] Figure 2 This is a schematic diagram of the overall high-throughput drug evaluation microfluidic chip in this invention.
[0026] Figure 3 This is a schematic diagram of the PDMS microvalve control layer structure in this invention;
[0027] Figure 4This is a schematic diagram of the PDMS thin film flow path chamber layer structure in this invention;
[0028] Figure 5 This is a schematic diagram of the PDMS microgroove layer structure in this invention;
[0029] Figure 6 This refers to a 3D cell sphere prepared according to one or more embodiments of the present invention.
[0030] Explanation of annotations in the diagram:
[0031] 1-PDMS microvalve control layer; 2-PDMS film flow path chamber layer; 3-PDMS microgroove layer; 4-lateral pressure valve; 5-longitudinal pressure valve; 6-lateral flow path; 7-longitudinal flow path; 8-drug inlet; 9-drug outlet; 10-cell inlet; 11-cell outlet; 12-culture chamber; 13-microgroove array; 14-lateral microvalve; 15-longitudinal microvalve; 16-liquid inlet and defoaming microcolumn; 17-drug inlet and defoaming chamber; 18-uniform microcolumn. Detailed Implementation
[0032] To further understand the content of the present invention, the present invention will be described in detail with reference to the embodiments. The following embodiments are used to illustrate the present invention, but are not intended to limit the scope of the present invention.
[0033] Reference Figure 1 and Figure 2 This embodiment relates to a high-throughput microfluidic chip for automated 3D cell culture and multi-dimensional drug efficacy evaluation, which includes, from top to bottom, a PDMS microvalve control layer 1, a PDMS thin film flow path chamber layer 2, and a PDMS microgroove layer 3;
[0034] The PDMS microvalve control layer 1 is provided with multiple lateral pressure valves 4 and multiple longitudinal pressure valves 5; the lateral pressure valves 4 and longitudinal pressure valves 5 are arranged in a cross pattern; the lateral pressure valves 4 and lateral pressure valves 5 are controlled to open and close by outputting pressure to the lower PDMS film flow path chamber layer 2.
[0035] The PDMS film flow path chamber layer 2 is provided with multiple transverse flow paths 6 and multiple longitudinal flow paths 7. The two ends of the transverse flow paths 6 are connected to drug inlet 8 and drug outlet 9, respectively, and the two ends of the longitudinal flow paths 7 are connected to cell inlet 10 and cell outlet 11, respectively. A culture chamber 12 is formed at the intersection of the transverse flow paths 6 and the longitudinal flow paths 7. The PDMS film flow path chamber layer 2 mainly provides inflow and outflow channels for cell and drug inlet, as well as a chamber for 3D cell culture.
[0036] The PDMS microgroove layer 3 contains a microgroove array 13 corresponding one-to-one with the culture chamber 12, which is used to provide high throughput for cell deposition and aggregation into 3D cell spheres. After the PDMS microvalve control layer 1, the PDMS thin film flow path chamber 2, and the PDMS microgroove layer 3 are prepared, they are synthesized into a high-throughput drug sieve microfluidic chip using oxygen plasma bonding.
[0037] like Figure 3 As shown, this is the PDMS microvalve control layer 1 in this embodiment. There are four transverse pressure valves 4 and five longitudinal pressure valves 5. The pressure inlets of both the transverse pressure valves 4 and the longitudinal pressure valves 5 are located on the upper surface of the PDMS microvalve control layer 1. Each pressure inlet of the transverse pressure valve 4 controls five transverse microvalves 14, and each pressure inlet of the longitudinal pressure valve 5 controls four longitudinal microvalves 15. The transverse microvalve 14 has a size of 3.1 x 1 mm, and the longitudinal microvalve 15 has a size of 4.1 x 1 mm. The control of the transverse microvalve 14 and the longitudinal microvalve 15 can be achieved by pneumatic or hydraulic pressure. The control pneumatic pressure is 300-500 mbar, and the longitudinal control valve pressure inlet 5 can control four longitudinal microvalves. The transverse and longitudinal microvalve 14 and the longitudinal microvalve 15 are arranged in an array structure. This embodiment includes a total of 20 transverse microvalve 14 and 20 longitudinal microvalve 15, located to the left and right and top and bottom of each culture chamber 12, respectively, to change the liquid flow path through the culture chamber 12.
[0038] like Figure 4 As shown, this is the PDMS film flow path chamber layer 2 in this embodiment. The PDMS film flow path chamber layer 2 has four longitudinal flow paths 7 for cell injection and four transverse flow paths 6 for drug injection, totaling 16 culture chambers 12. The inlet of the transverse flow path 6 is connected to the drug inlet 8 via the liquid inlet defoaming microcolumn 16 and the drug inlet defoaming chamber 17. The inlet of the longitudinal flow path 7 is connected to the cell inlet 10 via the cell homogenizing microcolumn 18. The inner diameter of the drug inlet 8 is 1 mm, the inner diameter of the drug outlet 9 is 1 mm, the inner diameter of the cell inlet 10 is 1 mm, the inner diameter of the cell outlet 11 is 1 mm, the inner diameter of the liquid inlet defoaming microcolumn 16 is 0.1 mm, the inner diameter of the cell homogenizing microcolumn 18 is 0.2 mm, the size of the culture chamber 12 is 2.7 x 2.7 mm, the width of the transverse flow path 6 is 0.3 mm, and the width of the longitudinal flow path 7 is 0.3 mm.
[0039] like Figure 5 The PDMS microgroove layer shown in this embodiment contains 16 microgroove arrays 13, each microgroove array 13 containing 19 microgrooves with a diameter of 0.25 mm and a depth of 200 mm.
[0040] Example 2
[0041] This embodiment relates to a method for fabricating a 3D cell-integrated array microfluidic chip for high-throughput drug evaluation, which includes two parts: mold processing and mold bonding.
[0042] The specific steps of mold processing include:
[0043] (1) Microvalve control layer: Clean a 4-inch silicon wafer with compressed air, then apply 2-3 ml of negative photoresist SU8-3025 to the center of the wafer, and achieve a coating thickness of 60 μm by swirl at 500 rpm for 10 seconds and 1200 rpm for 30 seconds. Pre-bake for 15 minutes on a hot plate at 95°C. Then move the wafer to the chuck of the UV exposure mask aligner and expose it through the control layer mask with a total energy deposition of 250 mJ. Then post-bake at 65°C for 1 minute and at 95°C for 5 minutes. Then immerse the wafer directly in SU-8 developer for about 8 minutes until a clear microstructure appears;
[0044] (2) Thin Film Flow Channel Chamber Layer: AZ40XT-11D photoresist was used, with spinning rates of 500 rpm for 10 s and 2000 rpm for 30 s to achieve a coating thickness of 35 μm. The silicon wafer was placed on a hot plate at 125°C for 7 minutes to complete the pre-baking step, and then the sample was exposed with a total energy of 400 mJ. Post-baking was performed at 105°C for 1 minute. Subsequently, the sample was allowed to stand at room temperature for 10 minutes before being directly immersed in AZ300MIF developer for development until the microstructure appeared. After drying, the sample was transferred to a hot plate and the microstructure was softened at 125°C for 3 minutes.
[0045] (3) Microgroove layer: Using SU8-2075 photoresist, a coating thickness of 220 μm was achieved at spin rates of 500 rpm for 10 s and 1200 rpm for 40 s. The silicon wafer was placed on a hot plate at 65°C for 7 minutes, and then on a hot plate at 95°C for 45 minutes to complete the pre-baking step. The sample was then exposed at an energy of 350 mJ. The subsequent post-baking step was baking at 65°C for 5 minutes and at 95°C for 15 minutes. Finally, the microgroove layer mold was fabricated by developing with SU8 developer for about 17 minutes.
[0046] The specific machining steps for mold bonding are as follows:
[0047] 44g of PDMS was prepared with a curing agent ratio of 10:1 and poured into a microvalve control layer mold. 30g of PDMS with the same curing agent ratio was poured into a microgroove layer mold. Then, 10g of PDMS with a curing agent ratio of 20:1 was poured into a thin film flow path chamber layer mold and spun at 500rpm for 10s and 1500rpm for 10s, respectively. For the microvalve control layer and microgroove layer, the PDMS, along with the mold, was placed in an oven at 80°C for 1.5h for complete curing. For the thin film flow path chamber layer, the PDMS, along with the mold, was placed in an oven at 80°C for 20 minutes for complete curing. After curing, the PDMS was peeled off from the three-layer mold and then perforated using a 0.75mm diameter punch. The three layers of PDMS were then bonded using oxygen plasma according to the previously designed configuration. After bonding is completed, a 0.6 x 0.9 mm steel needle is inserted into the drilled hole to complete the mold bonding of a drug sieve microfluidic chip.
[0048] In this embodiment, multiple cell types were cultured into 3D cell spheres. First, 1% Pluronic-F127 was introduced into the chip and then left to stand overnight at room temperature to complete the chip low-adhesion treatment, thereby promoting the aggregation of deposited cells into 3D cell spheres.
[0049] After washing the drug-filtering microfluidic chip with PBS, the microvalve was switched to longitudinal cell injection mode. Cultured intestinal cell line FHs 74Int, liver cell line THLE-2, cardiomyocyte cell line HL-1, and A549 cancer cell line were then prepared into cell suspensions. These suspensions were then injected into the chip using a syringe pump at a flow rate of 1 μL / m, allowing the cells to fall into the microchannels under gravity. After injection, the microvalve was switched to transverse flow mode, and culture medium was perfused at a flow rate of 10 μL / h.
[0050] After three days of perfusion culture, the cells aggregate into 3D cell spheroids, such as... Figure 6 As shown, a variety of 3D cell spheroids in vitro drug screening models simulating oral anticancer drugs were constructed using small intestine, liver, myocardium, and target lung cancer cells.
[0051] For the established 3D cell spheroid in vitro drug screening models, cisplatin, pemetrexed, and gemcitabine were used for 2 days of drug perfusion, followed by live-dead fluorescence staining to evaluate the side effects of various anticancer drugs on different organs and their efficacy against target cancer cells. Alternatively, different concentrations of the same drug were used to simultaneously test and evaluate the side effects and efficacy of different drug concentrations.
[0052] While the specific embodiments of the present invention have been described in detail above, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention, and modifications or variations without creative effort are still within the protection scope of the present invention.
Claims
1. A high-throughput microfluidic chip for automated 3D cell culture and multi-dimensional drug efficacy evaluation, characterized in that, From top to bottom, it consists of a PDMS microvalve control layer, a PDMS thin film flow path chamber layer, and a PDMS microgroove layer; The PDMS microvalve control layer is equipped with multiple lateral pressure valves and multiple longitudinal pressure valves; the lateral pressure valves and longitudinal pressure valves are arranged in a cross pattern; the lateral pressure valves and longitudinal pressure valves are controlled to open and close by outputting pressure to the lower PDMS film flow path chamber layer. The PDMS film flow path chamber layer is provided with multiple transverse flow paths and multiple longitudinal flow paths. The two ends of the transverse flow paths are connected to the drug inlet and the drug outlet, respectively, and the two ends of the longitudinal flow paths are connected to the cell inlet and the cell outlet, respectively. The intersection of the transverse and longitudinal flow paths forms a culture chamber. The inlet of the transverse flow path is connected to the drug inlet through the liquid inlet defoaming microcolumn and the drug inlet defoaming chamber in sequence, and the inlet of the longitudinal flow path is connected to the cell inlet through the cell homogenization microcolumn. The PDMS microgroove layer is a bottom layer structure independent of the PDMS thin film flow path chamber layer. It contains a microgroove array that corresponds one-to-one with the culture chamber, providing physical space for cell deposition and aggregation into 3D cell spheres.
2. The high-throughput microfluidic chip for automated 3D cell culture and multi-dimensional drug efficacy evaluation according to claim 1, characterized in that, The device has four transverse pressure valves and five longitudinal pressure valves. The pressure inlets of both the transverse and longitudinal pressure valves are located on the upper surface of the PDMS micro-valve control layer. Each transverse pressure valve consists of five transverse micro-valves, and each longitudinal pressure valve consists of four longitudinal micro-valves. The transverse micro-valve has a length of 1-4 mm and a width of 1-2 mm, while the longitudinal micro-valve has a length of 1-5 mm and a width of 1-2 mm.
3. The high-throughput microfluidic chip for automated 3D cell culture and multi-dimensional drug efficacy evaluation according to claim 1, characterized in that, The PDMS micro-valve control layer is controlled by input air pressure or hydraulic pressure.
4. The high-throughput microfluidic chip for automated 3D cell culture and multi-dimensional drug efficacy evaluation according to claim 1, characterized in that, The inner diameter of the drug inlet is 0.7-2 mm, the inner diameter of the drug outlet is 0.7-2 mm, the inner diameter of the cell inlet is 0.7-2 mm, the inner diameter of the cell outlet is 0.7-2 mm, the inner diameter of the defoaming microcolumn is 0.05-0.3 mm, the inner diameter of the cell homogenizing microcolumn is 0.1-0.3 mm, the dimensions of the culture chamber are 2-3 mm in length and 2-3 mm in width, the width of the transverse flow path is 0.1-1 mm, and the width of the longitudinal flow path is 0.1-1 mm.
5. The high-throughput microfluidic chip for automated 3D cell culture and multi-dimensional drug efficacy evaluation according to claim 1, characterized in that, Each microslot array consists of 19 microslots, with a diameter of 0.15-0.5 mm and a depth of 200-600 μm.
6. A method for preparing a high-throughput microfluidic chip for automated 3D cell culture and multi-dimensional drug efficacy evaluation as described in any one of claims 1-5, characterized in that, It includes the following steps: Fabrication of PDMS microvalve control layer: The silicon wafer is pretreated and cleaned with oxygen plasma. A layer of negative photoresist is spin-coated onto the cleaned silicon wafer. The silicon wafer with photoresist is vacuum-treated to remove air bubbles inside the photoresist. After removing air bubbles, the sample is placed on a hot plate for pre-baking. The pre-baked sample is placed in the sample tray of the UV exposure machine. The mask containing the microstructure pattern of the microvalve control layer is loaded onto the mask holder. The relative position of the sample and the mask is adjusted and the sample is raised to contact the mask before UV exposure. The UV-exposed samples were placed on a hot plate for post-baking. After baking, the sample is immersed in the developer until the microstructure of the microvalve appears, then the sample is cleaned. Pour the PDMS mixed solution into the silicon wafer mold prepared above, heat and solidify it, peel it off from the mold, cut it and punch holes in each control input port to obtain the PDMS micro valve control layer; PDMS thin film flow path chamber layer preparation: The silicon wafer is pretreated and cleaned with oxygen plasma, and positive photoresist is spin-coated on the cleaned silicon wafer. The silicon wafer with photoresist is vacuum-treated to remove air bubbles inside the photoresist. After removing air bubbles, the sample is placed on a hot plate for pre-baking. The pre-baked sample is placed in the sample tray of the UV exposure machine. The mask containing the microstructure pattern of the flow path chamber layer is loaded onto the mask holder. The relative position of the sample and the mask is adjusted and the sample is raised to contact the mask before UV exposure. The UV-exposed samples were placed on a hot plate for post-baking. After baking, the sample is immersed in the developer until the microstructure of the flow channel chamber appears, and then the sample is cleaned. The sample is placed on a hot plate machine at a suitable temperature to heat and soften the microstructure. The PDMS mixed solution is spun onto the prepared silicon wafer sample and heated to cure to obtain the PDMS thin film flow channel chamber layer. PDMS microgroove layer preparation: The silicon wafer is pretreated and cleaned with oxygen plasma. Negative photoresist is then spin-coated onto the cleaned silicon wafer. The silicon wafer with photoresist is vacuum-treated to remove air bubbles inside the photoresist. After removing air bubbles, the sample is placed on a hot plate for pre-baking. The pre-baked sample is placed in the sample tray of the UV exposure machine. The mask containing the microgroove layer microstructure pattern is loaded onto the mask holder. The relative position of the sample and the mask is adjusted and the sample is raised to contact the mask before UV exposure. The UV-exposed samples were placed on a hot plate for post-baking. After baking, the sample is immersed in the developer until the microstructure of the microgroove appears, then the sample is cleaned. The PDMS mixed solution is poured into the silicon wafer mold prepared by the above process, heated and cured, and then peeled off from the mold to obtain the PDMS microgroove layer; A high-throughput drug evaluation microfluidic chip is constructed by bonding multiple layers of PDMS: The PDMS microvalve control layer and the PDMS thin film flow path chamber layer still on the mold are simultaneously subjected to oxygen plasma treatment; the PDMS microvalve control layer and the PDMS thin film flow path chamber layer still on the mold are aligned and bonded, and then the two sample layers are peeled off from the mold and the inlet and outlet of the flow path chamber layer are perforated; the bonded sample and the PDMS microgroove layer are simultaneously subjected to oxygen plasma treatment, and then aligned and bonded.
7. The method for preparing a high-throughput microfluidic chip for automated 3D cell culture and multi-dimensional drug efficacy evaluation according to claim 6, characterized in that, Insert steel needles into the flow path inlet / outlet and the micro-valve control source inlet and connect them to flexible tubing.