A high-throughput tumor organoid culture system based on a pressure valve type microfluidic chip

Abstract

This invention discloses a high-throughput tumor organoid culture system based on a pressure-valve microfluidic chip, comprising: a microfluidic chip array consisting of multiple independently replaceable 96-well modules; a pneumatic control system including a gas source and pressure regulating device, a distributed valve drive board, and a main controller; an environmental control system including a temperature control module, a CO₂ control module, and a humidity control module; an imaging system including a distributed bright-field imaging module and a scanning high-resolution imaging module; and an AI control system including a cell detection and sorting module, a growth monitoring module, and an experimental execution module. The AI ​​control system receives image data acquired by the imaging system, identifies cell status in real time, and sends control commands to the pneumatic control system based on the identification results, automatically adjusting the on / off state of each pressure valve to achieve precise control of cell sorting, culture medium replacement, and drug treatment. This invention solves the problems of low throughput, poor reproducibility, and difficulty in implementing complex experimental designs in existing technologies.

Description

Technical Field

[0001] This invention belongs to the field of biochip and automated equipment technology, specifically relating to a high-throughput tumor organoid culture system based on a pressure valve microfluidic chip. Background Technology

[0002] Tumor organoids, as three-dimensional in vitro culture models, can effectively preserve the tissue structure and genetic characteristics of primary tumors, making them valuable for drug screening, personalized medicine, and tumor mechanism research. However, current organoid culture techniques mainly rely on manual manipulation, which presents the following problems: 1. Low throughput: Manual operation can only process dozens of samples at a time, which is difficult to meet the needs of high-throughput drug screening; 2. Poor repeatability: Large differences exist between different operators and different batches, affecting the reproducibility of the experiment; 3. High cost: It requires a lot of manpower and consumables, and the failure rate of cultivation is high; 4. Difficulty in implementing complex experimental designs: Complex experiments such as multiple concentration gradients and time series sampling are difficult to implement manually.

[0003] Microfluidic technology has made high-throughput cell culture possible, but existing microfluidic organoid culture platforms mostly use continuous flow or static culture methods, lacking flexible fluid control capabilities and making it difficult to perform complex operations such as culture medium replacement and drug treatment.

[0004] Pressure-valve microfluidics technology controls the opening and closing of fluid channels by driving thin film deformation with air pressure. It has advantages such as fast response speed, high control accuracy, and large-scale integration. However, its application in high-throughput organoid culture still faces the following challenges: 1. How to design a suitable chamber structure for organoid culture; 2. How to achieve large-scale integration of thousands of independent control units; 3. How to ensure stability and uniformity during long-term cultivation. Summary of the Invention

[0005] The purpose of this invention is to provide a high-throughput tumor organoid culture system based on a pressure valve microfluidic chip, so as to solve the problems of low throughput, poor reproducibility and difficulty in implementing complex experimental designs in the prior art.

[0006] The technical solution of the present invention is as follows: A high-throughput tumor organoid culture system based on a pressure-valve microfluidic chip, characterized in that it comprises: The microfluidic chip array consists of multiple independently replaceable 96-well modules, each module including: The culture chamber array is arranged in an 8×12 pattern, with each chamber having a diameter of 400-600 μm and a depth of 200-400 μm; The fluid network includes cell inlet, culture medium inlet, drug inlet, waste liquid outlet, and microchannels connecting the various chambers; The pressure valve array has an independent cell inlet valve, culture medium / drug inlet valve, and waste liquid outlet valve in each chamber. The air pressure control system includes: Gas source and pressure regulating device; Distributed valve drive board, each drive board controls 256 pressure valves; The main controller communicates with each driver board via real-time Ethernet; The main controller communicates with each drive board via real-time Ethernet to provide drive air pressure to the pressure valve array, thereby realizing independent control of the fluid flow in and out of each chamber. Environmental control systems, including: Temperature control module maintains the culture temperature at 37±0.5°C; The CO2 control module maintains a CO2 concentration of 5 ± 0.2%. Humidity control module maintains relative humidity >90%; This is used to maintain stable culture conditions in the environment where the microfluidic chip array is located.

[0007] Imaging system, including: Distributed bright-field imaging module, with each 96-aperture module configured with an independent CMOS camera; The scanning high-resolution imaging module can be moved to a designated module for confocal imaging; Used to acquire cell image data within the culture chamber in real time.

[0008] AI control system, including: The cell detection and sorting module identifies cells in real time and controls the pressure valve to achieve precise cell insertion. The growth monitoring module analyzes organoid morphological changes and provides early warnings of abnormalities; The experiment execution module automatically controls the fluid operation according to a preset plan.

[0009] The AI ​​control system receives image data collected by the imaging system, identifies cell status in real time, and sends control commands to the pneumatic control system based on the identification results. This automatically adjusts the on / off status of each pressure valve, enabling precise control of cell sorting, culture medium replacement, and drug processing.

[0010] Preferably, the culture chamber is cylindrical or inverted conical, with a micropillar array at the bottom to enhance substrate adhesion.

[0011] Preferably, the pressure valve adopts a three-layer structure: the upper layer is a pneumatic channel layer, the middle layer is a PDMS elastic film, and the lower layer is a fluid channel layer. The pneumatic pressure drives the film to deform, thereby opening and closing the fluid channel.

[0012] Preferably, the above-mentioned 96-hole module adopts a standardized interface, including: Fluid interfaces: cell inlet, culture medium inlet, drug inlet, waste liquid outlet; Pneumatic interface: A quick-plug interface for connecting to the valve drive board; Positioning interface: Mechanical positioning pins ensure installation accuracy.

[0013] Preferably, the system supports the parallel operation of 3,000 to 5,000 culture chambers, each of which can independently control the flow of fluid.

[0014] Preferably, the above-mentioned AI control system adopts a layered architecture, including: Edge layer: Each module is configured with an NVIDIA Jetson edge computing unit to run a lightweight AI model; it processes the image data acquired by the imaging system in real time and makes millisecond-level control decisions. Cloud layer: Runs deep learning models for growth prediction and drug response analysis.

[0015] Preferably, the imaging system described above includes an independent bright-field CMOS camera for each 96-well module and a movable confocal microscope.

[0016] Preferably, the response time of the pressure valve is less than 10ms and the driving air pressure is 30kPa.

[0017] Preferably, the culture chamber has a volume of 50-60 nL, a bottom micropillar diameter of 20 μm, a height of 20 μm, and a spacing of 50 μm.

[0018] A method for culturing tumor organoids based on the above system includes the following steps: A single-cell suspension of tumor cells is loaded into the system, and the AI ​​vision system identifies and controls the pressure valve to sort the cells into the cavity. The system automatically performs culture medium replacement, drug processing, and imaging monitoring; AI algorithms analyze organoid growth status and automatically adjust culture parameters; The system automatically performs endpoint detection and generates drug response curves.

[0019] The beneficial effects of this invention are: 1. High throughput: Supports parallel operation of 3,000-5,000 culture chambers, increasing throughput by more than 100 times compared to manual operation; 2. High uniformity: The pressure valve precisely controls the fluid operation of each chamber, with an inter-orifice CV value of <15%; 3. High degree of automation: The entire process, from cell seeding to endpoint testing, is automated, reducing manual intervention; 4. High flexibility: Each chamber is independently controlled, allowing for the flexible design of complex experimental schemes; 5. Scalability: Modular design allows for the addition or removal of modules as needed. Attached Figure Description

[0020] Figure 1 This is a schematic diagram of the overall system structure; Figure 2 This is a layout diagram of a microfluidic chip array; Figure 3 Top view of the 96-hole module; Figure 4 This is a cross-sectional view of the pressure valve structure; Figure 5 This is a fluid network topology diagram; Figure 6 This is a diagram of the air pressure control system architecture; Figure 7 This is a schematic diagram of an environmental control system. Figure 8 Diagram showing the configuration of the imaging system; Figure 9 This is a diagram of the AI ​​control system architecture. Figure 10 This is a flowchart of the experiment. Detailed Implementation

[0021] The present invention will be further described below with reference to the accompanying drawings. The following embodiments are only used to more clearly illustrate the technical solution of the present invention, and should not be used to limit the scope of protection of the present invention.

[0022] Example 1: Microfluidic Chip Design refer to Figure 2-5 The microfluidic chip in this embodiment adopts a modular design, with each module containing 96 culture chambers arranged in an 8×12 standard microplate layout.

[0023] The culture chamber is cylindrical, with a diameter of 500 μm, a depth of 300 μm, and a volume of approximately 59 nL. The bottom of the chamber contains an array of micropillars, each 20 μm in diameter and 20 μm in height, spaced 50 μm apart, to enhance the adhesion of the Matrigel matrix and prevent organoids from drifting during culture.

[0024] Each culture chamber is equipped with 3 independently controlled pressure valves: Cell inlet valve: controls the entry of single-cell suspension into the chamber; Culture medium / drug inlet valve: controls the entry of culture medium or drug solution; Waste liquid discharge valve: controls the discharge of waste liquid.

[0025] The pressure valve adopts a three-layer structure ( Figure 4 ): Upper layer: Pressure channel layer, containing pressure channels with a width of 200μm and a height of 50μm; Middle layer: PDMS elastic film, 20μm thick, used as valve actuator; Lower layer: Fluid channel layer, containing fluid channels with a width of 100μm and a height of 30μm, and valve seat structure.

[0026] When positive pressure (approximately 30 kPa) is applied to the air pressure channel, the PDMS diaphragm deforms downwards, pressing against the valve seat and closing the fluid passage; when the air pressure is released, the diaphragm elastically recovers, and the fluid passage opens. The valve response time is <10 ms, enabling precise fluid control.

[0027] Example 2: System Integration refer to Figure 1 , 6 -9, The system integration in this embodiment includes: The pneumatic control system adopts a distributed architecture. The main controller communicates with multiple valve actuator boards via EtherCAT real-time Ethernet, and each actuator board controls 256 pressure valves. The total number of valves in the system can reach more than 18,000, meeting the throughput requirements of 5,000 orifices.

[0028] The environmental control system adopts an incubator design, integrating heating, CO2, and humidification modules. It maintains stable culture conditions through PID control. Temperature control accuracy is ±0.5°C, and CO2 control accuracy is ±0.2%.

[0029] The imaging system employs a hybrid design: each 96-well module is equipped with an independent bright-field CMOS camera (2048×2048 pixels, 100fps) for real-time monitoring; the system is also equipped with a portable confocal microscope for high-resolution imaging and fluorescence detection.

[0030] The AI ​​control system adopts a layered architecture: Edge layer: Each module is configured with an NVIDIA Jetson edge computing unit to run lightweight AI models, enabling millisecond-level cell detection and valve control decisions; Cloud layer: Runs deep learning models and performs complex calculations such as growth prediction and drug response analysis.

[0031] Example 3: Application of Tumor Organoid Culture refer to Figure 10 This system was used for drug sensitivity testing of colorectal cancer organoids. Day 0: The digested tumor single-cell suspension was loaded into the system. The AI ​​vision system detected the cells in real time and controlled the pressure valves to sort the individual cells into each culture chamber. Approximately 5,000 cells were seeded into each chamber, with a seeding efficiency of >95%.

[0032] Day 1: After the matrix adhesive has cured, the system processes the drugs according to the preset protocol. This example tests the combined use of 5-FU and oxaliplatin, with 8 concentration gradients for each drug, 3 replicates, requiring a total of 192 chambers. The system automatically prepares the drug solution and distributes it to each chamber.

[0033] Days 2-14: The system automatically performs imaging monitoring every two days, and AI algorithms analyze changes in organoid area and morphology. When culture medium consumption or pH changes are detected, the system automatically replaces the culture medium.

[0034] Day 14: The system automatically performs endpoint detection, including live / dead staining imaging and ATP content determination, generates dose-response curves, and calculates IC50 values.

[0035] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the technical principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A high-throughput tumor organoid culture system based on a pressure valve microfluidic chip, characterized in that, The system comprises: a microfluidic chip array composed of multiple independently replaceable 96-well modules, each module comprising: an array of culture chambers arranged in 8x12, each chamber having a diameter of 400-600 μm and a depth of 200-400 μm; a fluid network including a cell inlet, a medium inlet, a drug inlet, a waste outlet, and microchannels connecting the chambers; an array of pressure valves, each chamber being equipped with independent cell inlet valves, medium / drug inlet valves, and waste outlet valves; a pneumatic control system including a gas source and pressure regulating device, distributed valve drive boards, and a main controller, which communicates with each drive board through real-time Ethernet, providing driving air pressure to the array of pressure valves, and achieving independent control of the fluid on-off of each chamber; an environmental control system including temperature control modules, CO2 control modules, and humidity control modules, for maintaining stable culture conditions in the environment of the microfluidic chip array; an imaging system including distributed bright-field imaging modules and scanning high-resolution imaging modules, for real-time acquisition of cell image data in the culture chambers; an AI control system including cell detection and sorting modules, growth monitoring modules, and experiment execution modules, which receives image data collected by the imaging system, identifies cell states in real time, and sends control instructions to the pneumatic control system according to the identification results, automatically adjusting the on-off state of each pressure valve, to achieve precise control of cell sorting, medium replacement, and drug treatment.

2. The high-throughput tumor organoid culture system based on a pressure valve microfluidic chip according to claim 1, characterized in that, The culture chambers are cylindrical or inverted conical, with a microcolumn array at the bottom to enhance Matrigel adhesion.

3. The high-throughput tumor organoid culture system based on a pressure valve microfluidic chip according to claim 1, characterized in that, The pressure valves adopt a three-layer structure: the upper layer is an air pressure channel layer, the middle layer is a PDMS elastic film, and the lower layer is a fluid channel layer. Air pressure drives the film to deform, achieving the opening and closing of the fluid channel.

4. The high-throughput tumor organoid culture system based on a pressure valve microfluidic chip according to claim 1, characterized in that, The 96-well module adopts a standardized interface, including: a fluid interface: cell inlet, medium inlet, drug inlet, and waste outlet; a pneumatic interface: a quick plug-in interface connected to the valve drive board; a positioning interface: mechanical positioning pins ensure installation accuracy.

5. The high-throughput tumor organoid culture system based on a pressure- valve microfluidic chip according to claim 1, characterized in that, The system supports the parallel operation of 3,000 to 5,000 culture chambers, with each chamber being independently controllable for fluid in-out.

6. The high-throughput tumor organoid culture system based on a pressure valve microfluidic chip according to claim 1, characterized in that, The AI control system adopts a layered architecture, including: an edge layer: each module is equipped with an NVIDIA Jetson edge computing unit, running lightweight AI models to process image data collected by the imaging system in real time and make millisecond-level valve control decisions; a cloud layer: running deep learning models for growth prediction and drug response analysis.

7. The high-throughput tumor organoid culture system based on a pressure- valve microfluidic chip according to claim 1, characterized in that, The imaging system includes a bright-field CMOS camera and a movable confocal microscope for each 96-well module.

8. The high-throughput tumor organoid culture system based on a pressure- valve microfluidic chip according to claim 1, characterized in that, The response time of the pressure valve is less than 10 ms, and the driving air pressure is 30 kPa.

9. The high-throughput tumor organoid culture system based on a pressure- valve microfluidic chip according to claim 1, characterized in that, The culture chamber has a volume of 50-60 nL, with a microcolumn diameter of 20 μm, a height of 20 μm, and a spacing of 50 μm.

10. A method of culturing tumor organoids based on the system of any one of claims 1-9, characterized in that, The system includes the following steps: loading tumor single-cell suspension into the system, and the AI vision system identifies and controls the pressure valve to sort cells into the chamber; the system automatically performs medium replacement, drug treatment, and imaging monitoring; AI algorithms analyze the growth state of the organoids and automatically adjust the culture parameters; The system automatically performs endpoint detection and generates a drug response curve.

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