Three-dimensional tumor cell culture platform and culture method based on micro-fluidic chip

Abstract

The invention belongs to but not limited to the technical field of microfluidics, and discloses a three-dimensional tumor cell culture platform and culture method based on a microfluidic chip. The gradient concentration dilution chip adopts an S-Tesla valve structure for active mixing, and gradient concentration dilution is formed on the fluid; the cell culture chip is used for culturing tumor cells; the programmable XY-axis moving holder controls the moving track of the XY-axis moving holder through programming, and the moving track of the holder is an S-shaped track; the micro peristaltic pump is used for driving liquid to flow and pumping drugs into the gradient concentration dilution chip and the cell culture chip; the microscope is used for monitoring the cell state in real time; and the AI data analysis platform is used for automatically processing the collected data, carrying out deep learning analysis and evaluating the growth state and drug response of the cells in real time. According to the invention, the error and workload of manual analysis are reduced, and the experiment efficiency and the data accuracy are improved.

Description

Technical Field

[0001] The present invention belongs to the field of microfluidics technology, and specifically relates to a three-dimensional tumor cell culture platform and culture method based on a microfluidics chip. The platform integrates microfluidics technology, automated control and artificial intelligence analysis to achieve three-dimensional culture, real-time monitoring and intelligent evaluation of tumor cells, and is suitable for fields such as cell biology research, drug screening and personalized medicine. Background Art

[0002] Traditional cell culture techniques mainly include flat plate culture and suspension culture. Flat plate culture is carried out on a two-dimensional plane, and cells grow in a monolayer. Although it is easy to operate, it cannot accurately simulate the growth and differentiation process of cells in the body due to the lack of spatial structure complexity. Suspension culture is suitable for certain specific cell types, but it is also difficult to simulate the three-dimensional microenvironment of in vivo tissues. Therefore, traditional cell culture methods have significant limitations in cell function research, drug screening and disease modeling.

[0003] With the deepening of biomedical research, three-dimensional cell culture (3D cell culture) has become a research hotspot. 3D cell culture can better simulate the in vivo environment, provide cell-cell interactions, cell-matrix interactions and complex cell arrangements, thereby improving the authenticity of cell physiological functions and drug responses. However, traditional three-dimensional culture methods (such as matrix-supported culture and spherical culture) have problems such as cumbersome operation, difficult culture environment control, high equipment requirements and long culture cycle.

[0004] The emergence of microfluidic chip technology provides a revolutionary solution for 3D cell culture. By precisely controlling the flow of tiny liquid streams, microfluidic chips can build a three-dimensional culture environment inside the chip and achieve precise nutrient delivery, waste removal, and oxygen supply. In addition, microfluidic chips also support high-throughput and diverse experimental designs while maintaining fine control over each tiny culture unit. Although microfluidic technology has made significant progress in cell culture, existing systems usually have problems such as low integration, complex operation, and insufficient real-time monitoring capabilities, which limit their application in high-throughput screening and personalized treatment.

[0005] Therefore, the prior art urgently needs to solve the following problems:

[0006] Complex operation: Traditional systems require independent operation training and high equipment requirements, and the process is cumbersome.

[0007] Inefficient data analysis: Lack of real-time data analysis and automated processing capabilities makes it difficult to meet high-throughput screening needs. Summary of the invention

[0008] In view of the problems existing in the prior art, the present invention provides a platform that integrates efficient mixing, gradient concentration dilution, cell culture, micro peristaltic pump delivery, and intelligent cell state monitoring and analysis. Through this platform, solution gradients can be efficiently generated to achieve accurate cell culture and state monitoring. Compared with traditional cell culture methods, this platform can not only provide a more physiologically relevant three-dimensional cell growth environment, but also achieve real-time cell state monitoring by integrating microscopy and fluorescent staining technology, avoiding the operational complexity and inefficiency of data analysis in traditional methods. In addition, the data in the platform is transmitted wirelessly or by hardware for artificial intelligence deep learning analysis, further improving the automation and intelligence level of data processing.

[0009] The present invention is implemented as follows: a three-dimensional tumor cell culture platform based on a microfluidic chip, wherein a numerically controlled microscope positioning frame and an XY axis moving platform are respectively arranged on the three-dimensional culture platform, a numerically controlled microscope is arranged on the upper end of the numerically controlled microscope positioning frame, the XY axis moving platform is located below the numerically controlled microscope, and a chip-pump fixing platform is arranged on the XY axis moving platform;

[0010] The upper end of the chip-pump fixing platform is provided with a glass cover, and the lower end of the glass cover is provided with an LED variable light wavelength lamp bead layer, and the variable light wavelength adjustment is realized by connecting the controller through the single-chip microcomputer; the cell culture chip is arranged on the upper end of the glass cover of the chip-pump fixing platform; a chip connection hose through hole is arranged on one side of the glass cover; a magnetic suction positioning hole is arranged at the bottom of the chip-pump fixing platform; a gradient concentration dilution chip fixing groove is arranged inside the chip-pump fixing platform for fixing the gradient concentration dilution chip; a peristaltic pump placement area is arranged at the bottom layer of the chip-pump fixing platform, a micropump fixing platform is arranged in the peristaltic pump placement area, a peristaltic pump and a centrifuge tube fixing frame are fixed on the micropump fixing platform, and the peristaltic pump can drive 4 fluid channels at the same time;

[0011] The peristaltic pump is provided with two channels, which are connected to the two channels on one side of the S-type Tesla valve (hereinafter referred to as "S-Tesla valve") of the gradient concentration dilution chip, so that the drug can be diverted by gradient concentration; the fluid channel on the other side of the gradient concentration dilution chip passes through the through hole of the chip connecting hose and out of the glass cover, and is connected to the cell culture chip through the hose.

[0012] Preferably, the gradient concentration dilution chip comprises a polydimethylsiloxane (PDMS) module and a glass sheet;

[0013] The polydimethylsiloxane module is provided with a first through hole, a second through hole, a third through hole, a fourth through hole, a fifth through hole, a sixth through hole, a seventh through hole, an eighth through hole, and a ninth through hole which penetrate through the thickness direction thereof, wherein the first through hole and the second through hole are sample injection holes of the gradient concentration dilution chip and are located on one side, and the remaining seven through holes are sample outlet holes and are located on the other side of the polydimethylsiloxane module;

[0014] The glass sheet is arranged below the polydimethylsiloxane module, the upper surface of the glass sheet and the lower surface of the polydimethylsiloxane module are bonded together through plasma technology, the hollow area on the lower surface of the polydimethylsiloxane module and the glass sheet constitute a microfluidic channel, the liquid to be diluted and the diluent are introduced into the first through hole and the second through hole, efficient active mixing is achieved through the S-Tesla valve structure, and the fluid is diluted to form a gradient concentration through the dilution model.

[0015] Preferably, the S-Tesla valve is an S-shaped structure; the S-Tesla valve has a width of 100 μm-200 μm; and a height of 100 μm-300 μm.

[0016] Preferably, the through hole is circular or elliptical.

[0017] Preferably, the cell culture chip comprises: a polydimethylsiloxane (PDMS) module and a glass sheet;

[0018] The polydimethylsiloxane module is provided with a sample liquid inlet and a sample liquid outlet which run through the thickness direction thereof and are respectively placed on both sides of the polydimethylsiloxane module, and is provided with a circular cell culture hole;

[0019] The glass sheet is arranged below the polydimethylsiloxane module, the upper surface of the glass sheet and the lower surface of the polydimethylsiloxane module are bonded together through plasma technology, and the hollow area on the lower surface of the polydimethylsiloxane module and the glass sheet constitute a cell culture chamber.

[0020] Preferably, the cell infusion channel is an S-shaped structure; the diameter of the cell culture hole is 200 μm-500 μm; and the height is 120 μm-500 μm.

[0021] Preferably, the cell culture well is cylindrical.

[0022] Preferably, the thickness of the polydimethylsiloxane module is 3 mm to 8 mm; and the thickness of the glass sheet is 0.5 mm to 2 mm.

[0023] Another object of the present invention is to provide a method for monitoring three-dimensional culture of tumor cells based on a three-dimensional culture platform of tumor cells using a microfluidic chip, comprising the following steps:

[0024] Step 1: Use a peristaltic pump to drive the drug to dilute the drug in a gradient concentration through a gradient concentration dilution chip, so that drug solutions of different concentrations pass through the cell culture chip;

[0025] Step 2: Observe the state of cells under fluorescence detection using a digitally controlled microscope by adjusting the LED lamp beads with variable wavelengths;

[0026] Step three: The data collected by the CNC microscope is transmitted wirelessly to the terminal, and the collected data is automatically processed using the AI ​​data analysis platform to perform deep learning analysis and evaluate the cell growth status and drug response in real time.

[0027] Another object of the present invention is to provide a three-dimensional tumor cell culture system based on a three-dimensional tumor cell culture platform of a microfluidic chip, comprising a chip module, a mechanical drive module, and a data analysis module;

[0028] The chip module includes a gradient concentration dilution chip and a cell culture chip; the gradient concentration dilution chip uses an S-Tesla valve structure to actively mix and dilute the fluid to form a gradient concentration; the cell culture chip is used to culture tumor cells;

[0029] The mechanical drive module includes a programmable XY-axis mobile platform and a micro peristaltic pump; the programmable XY-axis mobile platform controls the movement trajectory of the XY-axis mobile platform through programming, and the movement trajectory of the platform is an S-shaped trajectory; the micro peristaltic pump is used to drive the liquid flow to pump the drug into the chip module;

[0030] The data analysis module includes a CNC microscope and an AI data analysis platform; the CNC microscope is used to monitor the cell status in real time; the AI ​​data analysis platform is used to automatically process the collected data, perform deep learning analysis, and evaluate the cell growth status and drug response in real time.

[0031] In combination with the above technical solutions and the technical problems solved, the advantages and positive effects of the technical solutions to be protected by the present invention are as follows:

[0032] First, the present invention uses an S-Tesla valve structure to achieve active mixing in a microfluidic chip. The valve structure is specially designed to generate turbulence during fluid flow, allowing solutions to mix quickly and evenly. Compared with traditional methods that rely on passive mixing, the S-Tesla valve can achieve efficient mixing in a shorter channel length and form a stable and precise concentration gradient in the chip, thereby meeting the concentration control requirements for high-precision experiments.

[0033] The S-Tesla valve structure is designed to allow precise control of the flow rate of the fluid. By adjusting the geometric parameters inside the valve, the liquid presents a specific turbulent pattern when passing through the valve body. This mechanism not only ensures full mixing between the components, but also generates a stable gradient distribution according to experimental needs, thereby achieving controllable deployment of concentration gradients in the microfluidic chip and providing precise environmental variables for subsequent biological or chemical experiments.

[0034] The present invention highly integrates the cell culture chip, gradient concentration dilution chip, micro peristaltic pump and intelligent data analysis platform to form a unified platform. The platform accurately drives the liquid flow through a micro peristaltic pump, and a built-in microscope is used to monitor the cell state in real time, and LED variable light wavelength adjustment is used to achieve precise light control and fluorescence detection. The AI ​​data analysis system embedded in the platform performs automated deep learning processing on the collected images, fluid parameters and other signal data, achieving fast and accurate data analysis and real-time feedback, providing efficient data support for the experiment.

[0035] In terms of cell imaging detection, the present invention not only uses conventional live and dead cell dyes and reporter gene dyes, but also introduces fluorescent dyes with aggregation-induced emission (AIE) characteristics. This dye can significantly enhance the luminescent signal under specific conditions within or between cells, and eliminates the cleaning steps in traditional dye detection, making it suitable for long-term continuous monitoring of cell dynamics. This detection scheme works in conjunction with an integrated platform to achieve high-resolution imaging of cell states and long-term data acquisition, providing reliable data for real-time evaluation of cell growth and drug response.

[0036] Second, existing tumor cell culture methods mainly rely on two-dimensional culture, which cannot truly simulate the complex three-dimensional tumor microenvironment in the body, limiting the accuracy of the research. At the same time, existing drug concentration gradient generation technology usually requires external complex equipment, cumbersome operation, and insufficient precision in gradient control. In addition, traditional culture systems lack real-time monitoring methods, making it difficult to dynamically observe cell growth, morphological changes, and drug effects, limiting in-depth research on tumor cell behavior.

[0037] The present invention designs a three-dimensional tumor cell culture platform based on a microfluidic chip, and successfully constructs a three-dimensional culture microenvironment by integrating a gradient concentration dilution chip and a cell culture chip. The gradient concentration dilution chip adopts an S-Tesla valve structure to achieve efficient and accurate linear concentration gradient generation, providing more reliable data support for tumor cell drug screening. The culture platform has real-time monitoring and control functions through a CNC microscope and an LED variable wavelength lamp bead layer, and can dynamically observe tumor cell behavior and drug response processes.

[0038] The platform has a compact structure, and the modular design of the culture system is realized by connecting the peristaltic pump with the hose, which is easy to operate and has strong compatibility. The chip-pump fixing platform not only fixes the culture chip, but also optimizes the fluid connection layout of the drug and cell culture chamber, significantly improving the space utilization and flexibility of the culture system, and providing high adaptability for different experimental needs.

[0039] The present invention is not only suitable for three-dimensional culture of tumor cells, but can also be widely used in drug screening, tumor microenvironment research, personalized medicine and other fields, especially in anticancer drug research. By accurately simulating the tumor microenvironment and dynamically monitoring the effects of drugs, the present invention provides innovative technical support for tumor research, fills the gaps in the existing technology, and has significant application value and broad market prospects.

[0040] Third, the present invention integrates an efficient mixed gradient concentration dilution chip, a three-dimensional cell culture chip, a micro peristaltic pump and an intelligent data analysis platform, providing an efficient and automated experimental platform for cell culture and drug screening. Through a highly integrated design, the footprint and operational complexity of the experimental equipment are significantly reduced, while the use of microfluidics and intelligent analysis technology improves the accuracy and efficiency of the experiment. Compared with traditional experimental methods, the present invention realizes one-stop operation, which is suitable for multi-field applications such as drug screening, high-throughput experiments, disease modeling and personalized medicine, helping R&D institutions and enterprises to significantly save time and cost, and has broad commercial value.

[0041] The present invention uses a gradient concentration dilution chip designed with an S-Tesla valve to ensure that the generated concentration gradient is stable and uniform, eliminating errors in the manual dilution process. The three-dimensional cell culture chip provides culture conditions close to the in vivo environment, significantly improving the physiological relevance of experimental data. At the same time, the integrated AI analysis platform uses real-time data processing and deep learning algorithms to automatically exclude abnormal data and generate high-precision analysis results, greatly enhancing data reliability and experimental repeatability. In addition, the system replaces multiple independent devices with a single device, supports automated operation, reduces dependence on highly skilled personnel, and thus reduces equipment investment, labor costs, and consumption of experimental resources.

[0042] The traditional view is that gradient concentration dilution and cell culture need to be completed by independent equipment, which leads to complicated operation and reduced data consistency. The present invention highly integrates the dilution chip, culture chip and data analysis platform through innovative design, challenges the inherent concept of separation operation of traditional equipment, and realizes the organic combination of cross-domain technologies. At the same time, the present invention provides flexible optical adjustment function and efficient drug distribution method for drug screening experiments through peristaltic pump driven multi-channel microfluidic system and LED variable light wavelength lamp beads, further optimizing experimental efficiency and data reliability.

[0043] The present invention has important industrial application value in the field of tumor drug development and precision medicine. By simulating the three-dimensional microenvironment of tumors, the present invention significantly improves the clinical relevance of drug screening, reduces the probability of clinical trial failure, and accelerates the development of anti-tumor drugs, providing a scientific basis for tumor resistance research and treatment optimization. The highly integrated design reduces the complexity and cost of the equipment and improves the popularity of the platform. It is not only suitable for scientific research laboratories, but also can be used for industrial production needs, meeting the diverse application scenarios of drug screening, disease modeling, education and training, and providing innovative solutions and significant economic benefits for the development of the biotechnology and medical industries. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 It is a functional module diagram of a three-dimensional tumor cell culture platform based on a microfluidic chip provided in an embodiment of the present invention.

[0045] Figure 2 Schematic diagram of the structure of a three-dimensional tumor cell culture platform based on a microfluidic chip provided in an embodiment of the present invention;

[0046] Figure 3 It is a schematic diagram of the chip fixing platform module structure provided by an embodiment of the present invention, including various chip components.

[0047] Figure 4 It is a top view of a chip fixing platform provided by an embodiment of the present invention.

[0048] Figure 5 It is a bottom view of the chip fixing platform provided by an embodiment of the present invention.

[0049] Figure 6 It is a side view of a chip fixing platform provided by an embodiment of the present invention.

[0050] Figure 7 This is a gradient concentration chip diagram provided by an embodiment of the present invention.

[0051] Figure 8 This is a diagram of a cell culture chip provided in an embodiment of the present invention.

[0052] Fig. 9 It is a comparison diagram of the mixing efficiency of a COMSOL fluid simulation S-Tesla valve and an S-type T-type mixer provided in an embodiment of the present invention.

[0053] Fig.10 It is a physical picture and an assembly picture of a chip fixing platform provided in an embodiment of the present invention.

[0054] Fig.11 This is a physical picture of the chip provided by the embodiment of the present invention.

[0055] Fig.12This is a live and dead cell image in the chip provided by an embodiment of the present invention (green fluorescence is a live cell image, and red fluorescence is a dead cell image)

[0056] Fig.13 It is a flow chart of a method for monitoring three-dimensional culture of tumor cells of a three-dimensional culture platform of tumor cells based on a microfluidic chip provided in an embodiment of the present invention;

[0057] Fig.14 1. Models of three diluters provided in the embodiments of the present invention and simulation diagrams of mixing efficiency under different Reynolds numbers;

[0058] Fig.15 This is a simulation result diagram of mixing efficiency by selecting an equivalent mixing point for mixing at the same Reynolds number provided by an embodiment of the present invention;

[0059] Fig.16 is a gray value analysis diagram provided by an embodiment of the present invention;

[0060] Fig.17 is a comparison diagram of simulation curves provided by an embodiment of the present invention;

[0061] Fig.18 This is a diagram of cell states observed through a fluorescence microscope provided by an embodiment of the present invention.

[0062] In the figure: 1. CNC microscope positioning frame; 2. CNC microscope; 3. XY axis moving pan-tilt; 4. Chip-pump fixing platform; 5. Glass cover; 6. Chip connecting hose through hole; 7. LED variable wavelength lamp bead; 8. Magnetic positioning hole; 9. Gradient chip fixing groove; 10. Gradient concentration dilution chip; 11. Glass sheet of gradient concentration dilution chip (4cm*10cm); 12. Cell culture chip; 13. Glass sheet of cell culture chip; 14. Micropump fixing platform; 15. Centrifuge tube fixing frame; 16. First through hole; 17. Second through hole; 18. S-Tesla valve structure; 19. Sample outlet; 20. Sample liquid inlet; 21. Sample liquid outlet; 22. Cell culture chamber. DETAILED DESCRIPTION

[0063] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0064] The three-dimensional tumor cell culture platform based on microfluidic chip of the present invention integrates a numerical control microscope, an XY axis mobile platform, a chip-pump fixed platform and related microfluidic components and a light wave adjustment system. The platform realizes high-precision positioning and monitoring of the culture area through precise adjustment of the numerical control microscope positioning frame and the mobile platform. The chip and hose interface are set on the glass cover to facilitate fluid connection and sample observation. The bottom peristaltic pump drives the multi-channel fluid to realize the dynamic adjustment of drug gradient distribution and cell culture environment.

[0065] The peristaltic pump drives two channels to be connected to the S-Tesla valve inlet of the gradient concentration dilution chip 10, ensuring that the drug solution is distributed to different fluid channels according to the designed concentration gradient. The one-way fluid design of the S-Tesla valve avoids backflow and ensures accurate concentration distribution. Through this microfluidic control method, the platform realizes dynamic dilution and precise distribution of drugs in the culture environment.

[0066] The gradient concentration dilution chip is fixed in a dedicated slot on the chip-pump fixture. Its design enables automatic dilution of drug solutions and produces multiple fluid outputs of different concentrations. The gradient dilution chip diverts drug solutions into gradient solutions of different concentrations and outputs them to the cell culture chip through microfluidic channels, providing an accurate concentration environment for drug response experiments of tumor cells.

[0067] The output channel of the gradient concentration dilution chip is connected to the chip connection hose through hole of the glass cover through a hose, and then connected to the cell culture chip. A three-dimensional microenvironment is set up inside the cell culture chip for three-dimensional culture of tumor cells. Through this design, the drug solution can dynamically flow through the cell culture area to provide tumor cells with a microenvironment with different drug concentrations, thereby simulating the complex drug distribution in the body.

[0068] The cell culture chip is located on the top of the glass cover, and the LED variable wavelength lamp layer set below it adjusts the wavelength of the light wave through the single chip microcomputer and controller. This function can adjust the light wave range according to the experimental needs to meet the needs of specific wavelength light stimulation during cell culture, such as promoting specific metabolism or simulating optical treatment conditions.

[0069] The platform is comprehensively regulated by a CNC microscope and controller, which enables real-time observation and data recording, and dynamically adjusts drug gradient distribution and cell culture status. The coordinated work of the peristaltic pump, microfluidic chip, light regulation system and data analysis system makes the entire culture process efficient and automated, significantly improving the accuracy and repeatability of tumor cell culture and drug experiments.

[0070] The system consists of a chip module, a mechanical drive module, and a data analysis module. The chip module includes a gradient concentration dilution chip and a cell culture chip. The gradient concentration dilution chip uses an S-Tesla valve structure to achieve active mixing, while the cell culture chip is used for three-dimensional culture of tumor cells. The modules are interconnected through liquid channels and data transmission interfaces to form an integrated three-dimensional tumor cell culture platform.

[0071] The gradient concentration dilution chip is equipped with an S-Tesla valve structure, which generates turbulence during the liquid flow process and fully mixes the multi-path inlet liquid through active mixing to form a predetermined gradient concentration. The fluid in the mixing process gradually adjusts the concentration along the fixed flow path inside the chip to achieve a continuous change from high concentration to low concentration, providing precise concentration gradient conditions for cell culture.

[0072] The cell culture chip is designed as a multi-cavity structure, and its inner surface is microstructured to adapt to the attachment and growth of tumor cells in a three-dimensional environment. The gradient concentration dilution chip is connected to the cell culture chip through a flow channel, so that the liquid with gradient concentration after mixing can be evenly injected into each culture cavity, thereby forming multiple preset concentration areas in the chip, providing a diverse growth environment for cell culture.

[0073] The mechanical drive module includes a programmable XY-axis mobile platform and a micro peristaltic pump. The programmable XY-axis mobile platform is driven by an embedded controller and accurately positions the chip module according to the preset S-shaped motion trajectory to ensure that the liquid injection position and timing meet the design requirements. The precise movement of the platform ensures that during the entire injection process, each area can obtain the expected liquid distribution of the corresponding concentration.

[0074] The micro peristaltic pump achieves quantitative liquid delivery by periodically compressing and releasing the flexible pipe, and its operation is precisely controlled by the control unit. The output liquid of the pump enters the chip module at a stable flow rate, coordinating with the S-shaped movement of the pan / tilt table to evenly distribute the drug or culture medium in the gradient concentration dilution chip and cell culture chip, thereby achieving continuous and accurate liquid supply.

[0075] The data analysis module includes a CNC microscope and an AI data analysis platform. The CNC microscope collects high-resolution images in the cell culture chip in real time and transmits the image data to the AI ​​data analysis platform through a high-speed interface. The platform has an embedded deep learning algorithm to automatically process and extract features from the collected images, and quantitatively analyze cell growth status and drug response. The processed data is output in digital and graphical form, providing accurate data support for subsequent parameter adjustments and research on the platform.

[0076] like Figure 1As shown, an embodiment of the present invention provides a tumor cell three-dimensional culture system based on a tumor cell three-dimensional culture platform of a microfluidic chip, including a chip module, a mechanical drive module, and a data analysis module;

[0077] The chip module includes a gradient concentration dilution chip 10 and a cell culture chip 12; the gradient concentration dilution chip 10 uses an S-Tesla valve structure to actively mix and dilute the fluid to form a gradient concentration; the cell culture chip 12 is used to culture tumor cells;

[0078] The mechanical drive module includes a programmable XY-axis mobile platform and a micro peristaltic pump; the programmable XY-axis mobile platform controls the movement trajectory of the XY-axis mobile platform through programming, and the movement trajectory of the platform is an S-shaped trajectory; the micro peristaltic pump is used to drive the liquid flow to pump the drug into the chip module;

[0079] The data analysis module includes a numerically controlled microscope 2 and an AI data analysis platform; the numerically controlled microscope 2 is used to monitor the cell status in real time; the AI ​​data analysis platform is used to automatically process the collected data, perform deep learning analysis, and evaluate the cell growth status and drug response in real time.

[0080] like Figure 2 As shown, an embodiment of the present invention provides a three-dimensional tumor cell culture platform based on a microfluidic chip, wherein a numerically controlled microscope positioning frame 1 and an XY axis moving platform 3 are respectively provided on the three-dimensional culture platform, a numerically controlled microscope 2 is provided on the upper end of the numerically controlled microscope positioning frame 3, the XY axis moving platform 3 is located below the numerically controlled microscope 2, and a chip-pump fixing platform 4 is provided on the XY axis moving platform 3;

[0081] like Figure 3 As shown, a glass cover 5 is arranged at the upper end of the chip-pump fixing platform 4, and an LED variable light wavelength lamp bead layer is arranged at the lower end of the glass cover 5, and the variable light wavelength adjustment is realized by connecting the controller through the single chip microcomputer; the cell culture chip 12 is arranged at the upper end of the glass cover 5 of the chip-pump fixing platform 4; a chip connection hose through hole 6 ( Figure 4 ); The bottom of the chip-pump fixing platform 4 is provided with a magnetic positioning hole 8 ( Figure 5 ); the chip-pump fixing station 4 is provided with a gradient concentration dilution chip fixing groove 9 ( Figure 6 ), used to fix the gradient concentration dilution chip 10; the bottom layer of the chip-pump fixing platform 4 is provided with a peristaltic pump placement area, and a micropump fixing platform 14 is provided in the peristaltic pump placement area. A peristaltic pump and a centrifuge tube fixing frame 15 are fixed on the micropump fixing platform 14. The peristaltic pump can drive four fluid channels at the same time;

[0082] The peristaltic pump is provided with two channels, which are connected to the two channels on one side of the S-Tesla valve of the gradient concentration dilution chip 10, so that the drug can be diverted by gradient concentration; the fluid channel on the other side of the gradient concentration dilution chip 10 passes through the glass cover 5 through the chip connection hose through hole 6, and is connected to the cell culture chip 12 through the hose.

[0083] like Figure 7 As shown, the gradient concentration dilution chip 10 includes a polydimethylsiloxane (PDMS) module and a glass sheet;

[0084] The polydimethylsiloxane module is provided with a first through hole 16, a second through hole 17, a third through hole, a fourth through hole, a fifth through hole, a sixth through hole, a seventh through hole, an eighth through hole, and a ninth through hole which penetrate through the thickness direction thereof. The first through hole 16 and the second through hole 17 are sample injection holes of the gradient concentration dilution chip and are located on one side. The remaining seven through holes are sample outlet holes and are located on the other side of the polydimethylsiloxane module.

[0085] The glass sheet is arranged below the polydimethylsiloxane module, the upper surface of the glass sheet and the lower surface of the polydimethylsiloxane module are bonded together through plasma technology, the hollow area on the lower surface of the polydimethylsiloxane module and the glass sheet constitute a microfluidic channel, the liquid to be diluted and the diluent are introduced into the first through hole and the second through hole, efficient active mixing is achieved through the S-Tesla valve structure, and the fluid is diluted to form a gradient concentration through the dilution model.

[0086] Preferably, the S-Tesla valve is an S-shaped structure; the S-Tesla valve has a width of 100 μm-200 μm; and a height of 100 μm-300 μm.

[0087] Preferably, the through hole is circular or elliptical.

[0088] like Figure 8 As shown, the cell culture chip 12 includes: a polydimethylsiloxane (PDMS) module and a glass sheet;

[0089] The polydimethylsiloxane module is provided with a sample liquid inlet 20 and a sample liquid outlet 21 which penetrate through the thickness direction thereof and are respectively disposed on both sides of the polydimethylsiloxane module, and a circular cell culture hole is provided;

[0090] The glass sheet is disposed below the polydimethylsiloxane module, the upper surface of the glass sheet is bonded to the lower surface of the polydimethylsiloxane module through plasma technology, and the hollow area on the lower surface of the polydimethylsiloxane module and the glass sheet constitute a cell culture chamber 22 .

[0091] Preferably, the cell infusion channel is an S-shaped structure; the diameter of the cell culture hole is 200 μm-500 μm; and the height is 120 μm-500 μm.

[0092] Preferably, the cell culture well is cylindrical.

[0093] Preferably, the thickness of the polydimethylsiloxane module is 3 mm to 8 mm; and the thickness of the glass sheet is 0.5 mm to 2 mm.

[0094] like Fig.13 As shown, an embodiment of the present invention provides a method for monitoring three-dimensional culture of tumor cells based on a three-dimensional culture platform of tumor cells using a microfluidic chip, comprising the following steps:

[0095] Step 1: Use a peristaltic pump to drive the drug to dilute the drug in a gradient concentration through the gradient concentration dilution chip 10, so that drug solutions of different concentrations pass through the cell culture chip 12;

[0096] Step 2, by adjusting the LED lamp beads with variable wavelength, using a digital control microscope 2 to observe the state of the cells under fluorescence detection;

[0097] Step three: The data collected by the CNC microscope 2 is transmitted wirelessly to the terminal, and the collected data is automatically processed using the AI ​​data analysis platform to perform deep learning analysis and evaluate the cell growth status and drug response in real time.

[0098] Example 1: A three-dimensional cell culture and artificial intelligence-assisted automated data analysis method based on a microfluidic chip, comprising the following steps:

[0099] (1) Fabrication of gradient concentration chip:

[0100] like Figure 7 As shown, the microfluidic chip used in this embodiment has two layers, the bottom layer is made of glass sheet material, and the upper layer is made of polydimethylsilane (PDMS) material.

[0101] The chip was made using the emerging 3D printing process. First, the modeling software Solidworks (2019b) was used for modeling. The microchannel was 120 μm wide and 240 μm high. A positive mold model of the active mixing model of the S-Tesla valve was designed and the file format was exported as STL. The Chitubox slicing software was used to import the obtained STL file and slice it to obtain a .goo file. The commercial MSLA printer, Elegoo Mars4Ultra, was used to print the positive mold. The obtained positive mold was cleaned with anhydrous ethanol and then fully cured under a 405nm ultraviolet post-curing lamp for 8 hours. PDMS and cross-linking agent were prepared and fully mixed in a mass ratio of 10:1. The mixture was poured onto the printed positive mold. The bubbles in the mold were removed by vacuum treatment. After heating at 80°C for 10 minutes, the PDMS was cured and formed. After the PDMS layer was taken out, the required joint parts were punched. After that, the chips and glass sheets were plasma treated (86W, 65s). The chips and glass sheets were taken out, and they were quickly bonded and reinforced on a hot stage at 80°C for 5 minutes. A gradient concentration chip was obtained. The seven sample outlets with different concentrations in the figure are used to connect the cell culture chip, so as to construct a cell culture scheme under different concentration gradients.

[0102] (2) Fabrication of cell chips

[0103] like Figure 8 As shown, the microfluidic chip used in this embodiment has two layers, the bottom layer is made of glass sheet material, and the upper layer is made of polydimethylsilane (PDMS) material.

[0104] The chip was made using the emerging 3D printing process. First, the modeling software Solidworks (2019b) was used for modeling. The microchannel was 300 μm wide and 180 μm high, and the microwell array was a cylindrical array with a diameter of 400 μm and a height of 400 μm. The exported file format was STL. The Chitubox slicing software was used to import the obtained STL file, and the .goo file was obtained by slicing. The commercial MSLA printer, Elegoo Mars4 Ultra, was used to print the positive mold. The obtained positive mold was cleaned with anhydrous ethanol and fully cured under a 405nm ultraviolet post-curing lamp for 8 hours. PDMS and cross-linking agent were prepared and fully mixed in a mass ratio of 10:1, and then poured onto the printed positive mold. The bubbles in the mold were removed by vacuum treatment. After heating at 80°C for 10 minutes, PDMS was cured and formed. After the PDMS layer was taken out, the required joint part was punched, and then plasma treatment (86W, 65s) was performed together with the glass sheet. The chip and glass sheet were taken out, quickly attached, and reinforced on a hot stage at 80°C for 5 minutes. A cell culture chip was obtained.

[0105] (3) Preparation of cell / matrigel mixture and cell experiment steps

[0106] Weigh 5 g of gelatin powder, add it into a round-bottom flask, add 50 ml of water and heat until completely dissolved; weigh 8 ml of methacrylamide, slowly add dropwise to the gelatin solution, react for 3 hours, take an equal amount of PBS and add it to the reaction solution to terminate the reaction, dialyze and freeze-dry to obtain pure GelMa, prepare 10% w / v GelMa solution and set aside.

[0107] MCF-7 breast cancer tumor cells were used for microfluidic chip cell culture. Before the cell experiment, all instruments were sterilized by high pressure, disinfected with 75% alcohol, and sterilized by ultraviolet for 2 hours. The matrix gel was sterilized by 0.22μm filtration. The specific steps of the cell experiment are as follows:

[0108] ① When the cells grow to more than 90% of the T25 culture flask, discard the culture medium and add sterile PBS to wash once.

[0109] ② After trypsin digestion, centrifuge at 1000 rpm for 5 minutes

[0110] ③After centrifugation, discard the supernatant, add sterilized matrix gel, count, and dilute the cells to 1×106 cells / ml

[0111] ④ Pass the cell / matrigel mixed solution into the chip, use fluorinated oil to push out the matrix gel mixed solution in the channel, and then irradiate with 365nm UV lamp for 30s

[0112] ⑤ Pass sterile cell culture medium, and fix the culture medium and drug-loaded culture medium through the culture medium support 15 to study the effects of different concentrations of drugs on tumor cells.

[0113] (4) Capture and analysis of cell survival status

[0114] The movement trajectory of the XY pan-tilt table 3 and the timed photography of the microscope are controlled by programming. The movement trajectory of the pan-tilt table is an S-shaped trajectory, which is the same as the layout of the cell culture chip. A round of photography is set every 4 hours, and the interval between each round of photography is 5 seconds. The lateral movement distance of the pan-tilt table is 750μm. After each round of photography, it resets and waits for the next round of photography. If fluorescence images need to be taken, before each round of photography, the LED ultraviolet lamp beads with the required wavelength are embedded and turned on.

[0115] (5) Data processing

[0116] The pictures taken by the microscope can be transmitted wirelessly to the terminal, which is equipped with a deep learning data processing system and an integrated automatic recognition algorithm to automatically analyze the tumor boundaries. The growth characteristics and invasiveness of the tumor can be described by digitally analyzing the tumor size.

[0117] 1. Specific application fields or related applications of the present invention.

[0118] 1. Drug screening and high-throughput experiments

[0119] a) In a gradient concentration dilution chip, drug solution and diluent are mixed through an S-Tesla valve structure to generate five anticancer drug solutions of different concentrations (eg, 10 μM, 1 μM, 0.1 μM, 0.01 μM, 0.001 μM).

[0120] b) The diluted drug solution is injected into a three-dimensional cell culture chip through a peristaltic pump, and tumor cells (such as breast cancer cells MCF-7) are pre-inoculated in the culture chip.

[0121] c) Through the LED variable light wavelength lamp bead layer, select specific fluorescence wavelengths (such as 488nm and 561nm) to observe cell apoptosis under the action of drugs, and use fluorescence to label apoptosis-related proteins (such as Caspase-3).

[0122] d) Collect microscopic images in real time, calculate the apoptosis rate of cells under different drug concentrations through the AI ​​analysis module, and draw the dose-effect curve.

[0123] 2. Personalized medicine and precision treatment

[0124] a) Obtain biopsy tissue from the patient and obtain tumor cell suspension through enzymatic hydrolysis and cell separation techniques.

[0125] b) Patient-derived tumor cells were inoculated into the cell culture chip, and the patient's serum was introduced into the microfluidic channel to simulate the in vivo microenvironment.

[0126] c) The gradient dilution chip generates different concentrations of chemotherapy drugs (such as paclitaxel: 50 μM, 25 μM, 12.5 μM, 6.25 μM, 3.125 μM) and injects them into the culture chip.

[0127] d) Monitor cell proliferation and apoptosis through microscopy and fluorescent labeling technology, and record the cell status after drug action in real time.

[0128] e) Import the data into the AI ​​analysis platform to screen out the drug concentration with the best inhibitory effect on the patient's tumor cells and the lowest toxicity.

[0129] 3. Disease modeling and mechanism research

[0130] a) Mouse primary glioma cells were inoculated into the 3D cell culture chip and cultured for 7 days to simulate the tumor microenvironment and promote tumor cell growth and cell-cell interaction.

[0131] b) Using a gradient concentration dilution chip to generate a concentration gradient of a chemotherapeutic drug (such as temozolomide) (such as 100 μM, 50 μM, 25 μM, 10 μM, 5 μM), and continuously injecting it into the tumor cell culture chamber to simulate the diffusion and effect of the drug in the tumor tissue.

[0132] c) Use fluorescent labeling technology (such as labeling the proliferation marker Ki-67 and the apoptosis marker Caspase-3) to observe the dynamic changes of tumor cell proliferation and apoptosis in real time to evaluate the effect of chemotherapy drugs on tumor cells.

[0133] d) Collect experimental data, quantify the proliferation rate, apoptosis rate and cell morphology changes of tumor cells through the AI ​​analysis platform, analyze the inhibitory effects of different concentrations of chemotherapy drugs on tumor cell growth, and evaluate their anti-tumor activity.

[0134] 4. Automated monitoring of the synergistic inhibitory effects of multi-component combinations of traditional Chinese medicine on tumor cells through microfluidics technology

[0135] a) Extracting different effective parts of the target Chinese medicine, such as total flavonoids, total alkaloids, triterpenoid saponins, etc., and dissolving them into a stock solution of a determined concentration.

[0136] b) Using a gradient concentration dilution chip, the different components of the above three traditional Chinese medicines are automatically generated into multiple groups of compatibility ratios according to a certain ratio (such as 1:1:1, 2:1:1, 1:2:1, 1:1:2, etc.), and different concentration gradients (such as high, medium, and low concentrations) are generated for each group of compatibility.

[0137] c) Inoculate a human tumor cell line (such as liver cancer cell HepG2 or lung cancer cell A549) into the cell culture chip and culture it to the logarithmic growth phase.

[0138] d) The multi-fraction solution of traditional Chinese medicine generated by the dilution chip is automatically injected into the cell culture chamber, and the automated delivery of different concentrations and combinations is achieved through the microfluidic system.

[0139] e) Observe cell proliferation, apoptosis and activity changes through LED variable wavelength fluorescence microscopy system; use fluorescent probes to label apoptosis-related molecules (such as Bax, Bcl-2 or Caspase-3).

[0140] f) The experimental data is automatically analyzed through the AI ​​platform to quantify the synergistic index (CI) of different concentrations and combinations, and to screen out the optimal compatibility ratio and concentration range.

[0141] 2. Relevant evidence of the technical effects obtained by the embodiments of the present invention.

[0142] 1. Overview of the technical effects of gradient concentration generation ( Fig.14)

[0143] like Fig.14 As shown in the figure, three diluter models (including S-Tesla valve) were simulated and analyzed by COMSOL 5.4, and their mixing efficiency was compared under different Reynolds numbers. The results show that the S-Tesla valve structure has a significant mixing optimization effect under the same Reynolds number conditions, especially under low Reynolds number conditions.

[0144] 2. Effect of Reynolds number on mixing efficiency ( Fig.15 )

[0145] Fig.15 The simulation results of mixing efficiency at the same Reynolds number are shown. After comparing the equivalent mixing points, it is found that the S-Tesla valve structure not only has a shorter mixing path, but also significantly improves the mixing efficiency. Its unique geometric design effectively optimizes the microfluidic mixing process and provides support for chip spatial layout and efficiency improvement.

[0146] 3. Concentration gradient linearity verification ( Fig.16 and Fig.17 )

[0147] Experimental verification using methylene blue solution ( Fig.16 ), different concentration gradient solutions were generated by chip and gray value analysis was performed. The experimental results showed a linear trend that was highly consistent with the simulation curve ( Fig.17 ). This result shows that the designed gradient dilution chip can generate accurate and stable linear concentration gradients and is suitable for a variety of biological experimental needs.

[0148] 4. Technical effects of three-dimensional cell culture ( Fig.18 Left)

[0149] like Fig.18 As shown on the left, breast cancer cells were inoculated in the 3D chip culture chamber, and anticancer drugs with different concentration gradients were continuously provided during the culture process. The results showed that the system effectively simulated the tumor microenvironment and could realize dynamic monitoring of tumor cells under multi-gradient conditions.

[0150] 5. Real-time monitoring of fluorescent labeling ( Fig.18 right)

[0151] In real-time observation, apoptosis-related proteins (such as Caspase-3) were fluorescently labeled, and drug-induced cell apoptosis was observed to be concentration-dependent by fluorescence microscopy ( Fig.18 Right). The fluorescence signal in the high-concentration area is significantly enhanced, indicating that the drug's apoptotic effect on tumor cells increases with increasing concentration.

[0152] 6. Quantitative analysis and application of experimental data ( Fig.18Combined with data analysis)

[0153] Fig.18 Combined with the AI ​​platform to quantify and analyze data, including cell survival rate, apoptosis rate and cell morphology changes, the mechanism of action of anticancer drug concentration gradient on tumor cells was further verified. This technology provides accurate data support and experimental basis for the practical application of microfluidic chips in drug screening and anti-tumor research.

[0154] The above description, combined with the figure numbers, elaborates on the experimental content and results, and emphasizes the unique technical effects of the S-Tesla valve and the three-dimensional culture chip. The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any modification, equivalent substitution and improvement made by any technician familiar with the technical field within the technical scope disclosed by the present invention and within the spirit and principle of the present invention shall be covered within the protection scope of the present invention.

Claims

1. A three-dimensional tumor cell culture platform based on a microfluidic chip, characterized in that: include: CNC microscope positioning frame, XY axis moving platform, chip-pump fixing platform; A numerically controlled microscope is arranged on the numerically controlled microscope positioning frame, an XY axis movable platform is located below the numerically controlled microscope, and the chip-pump fixing platform is arranged on the XY axis movable platform; A glass cover is provided at the upper end of the chip-pump fixing platform, and a chip connection hose through hole is provided at one side of the glass cover; The chip-pump fixing platform is provided with a gradient concentration dilution chip fixing groove inside, and the gradient concentration dilution chip is fixed in the fixing groove; A peristaltic pump placement area is arranged at the bottom of the chip-pump fixing platform, a micro pump fixing platform is arranged in the peristaltic pump placement area, and a peristaltic pump is fixed on the micro pump fixing platform; The peristaltic pump is connected to the gradient concentration dilution chip through at least two channels; The gradient concentration dilution chip is connected to the cell culture chip through a hose, and the cell culture chip is arranged on the upper end of the glass cover.

2. The three-dimensional tumor cell culture platform based on a microfluidic chip according to claim 1, characterized in that: The gradient concentration dilution chip comprises a polydimethylsiloxane module and a glass sheet; The polydimethylsiloxane module is provided with a plurality of through holes extending through the thickness direction thereof, at least two of which are used as sample inlet holes, and the rest are sample outlet holes; The glass sheet is arranged below the polydimethylsiloxane module. The glass sheet and the polydimethylsiloxane module are bonded together by plasma technology. The hollow area on the lower surface of the module and the glass sheet together form a microfluidic channel.

3. The three-dimensional tumor cell culture platform based on a microfluidic chip according to claim 2, characterized in that: The gradient concentration dilution chip is provided with an S-Tesla valve with an S-shaped structure; the width of the S-Tesla valve is 100 μm-200 μm, and the height is 100 μm-300 μm.

4. The three-dimensional tumor cell culture platform based on a microfluidic chip according to claim 2, characterized in that: The through hole is in a circular or elliptical shape.

5. The three-dimensional tumor cell culture platform based on a microfluidic chip according to claim 1, characterized in that: The cell culture chip comprises a polydimethylsiloxane module and a glass sheet; The polydimethylsiloxane module is provided with a sample liquid inlet and a sample liquid outlet extending through the thickness direction thereof, and located on both sides of the module, and a circular cell culture hole is provided inside the module; The glass sheet is arranged below the polydimethylsiloxane module and bonded together by plasma technology; The hollow area on the lower surface of the polydimethylsiloxane module and the glass sheet together form a cell culture chamber.

6. The three-dimensional tumor cell culture platform based on a microfluidic chip according to claim 5, characterized in that: The cell culture hole has a diameter of 200 μm-500 μm and a height of 120 μm-500 μm.

7. The three-dimensional tumor cell culture platform based on a microfluidic chip according to claim 5, characterized in that: The cell culture well is cylindrical; the thickness of the polydimethylsiloxane module is 3mm-8mm, and the thickness of the glass sheet is 0.5mm-2mm.

8. A three-dimensional tumor cell culture method based on a microfluidic chip, characterized in that: The following steps are involved: Fix the cell culture chip on the chip-pump fixing platform and connect it to the gradient concentration dilution chip through a hose; The liquid to be diluted and the diluent are introduced into the injection holes of the gradient concentration dilution chip through a peristaltic pump, and the fluids are mixed and diluted through the S-Tesla valve in the gradient dilution chip to generate a linear concentration gradient; The generated concentration gradient solution is transported to the liquid inlet of the cell culture chip through a hose to culture the tumor cells.

9. The three-dimensional tumor cell culture method based on a microfluidic chip according to claim 8, characterized in that: The following steps are also included: Inoculating tumor cells in a cell culture chamber for a culture period of 1-7 days; The light wavelength is adjusted by the LED variable light wavelength lamp bead layer to achieve control of the cell microenvironment during the culture process; A digitally controlled microscope was used to observe the cell status in real time during the culture process and to record the cell growth and morphological change data.

10. The three-dimensional tumor cell culture method based on a microfluidic chip according to claim 8, characterized in that: By adjusting the geometric parameters of the S-Tesla valve in the gradient concentration dilution chip, including width and height, the mixing efficiency of the dilution liquid and the diluent is controlled to form a variety of solutions with different concentration gradients to act on tumor cells.

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