Micro-column chip as well as preparation method and use method thereof

Abstract

The invention discloses a microcolumn chip as well as a preparation method and a use method thereof, and relates to the technical field of biological chips. The microcolumn chip comprises a glass substrate and a PDMS chip arranged on one side of the glass substrate, a cell migration and deformation channel is arranged in the PDMS chip, and a plurality of microcolumns are arranged in the cell migration and deformation channel; the device further comprises a cell injection channel, the downstream of the cell injection channel is communicated with a cell calibration channel, and the cell calibration channel is communicated with the cell migration and deformation channel; the downstream of the Christmas tree-shaped channel is communicated with the cell migration and deformation channel, and the upstream of the Christmas tree-shaped channel is communicated with multiple groups of serpentine channels; according to the microcolumn chip, through the coupling design of the microcolumn channel and the stable chemical gradient, mechanical extrusion and chemotactic signals can be synchronously applied on the basis of simulating the real microenvironment of cells, high bionic simulation of single cell migration and deformation behaviors is achieved, and the limitation that an existing chip can only independently simulate the chemical gradient or mechanical compression is broken through.

Description

Technical Field

[0001] This invention relates to the field of biochip technology, specifically to a micropillar chip and its preparation and usage methods. Background Technology

[0002] Microfluidic chips, also known as lab-on-a-chip, are a core technology for manipulating fluids within a micrometer-scale space. This technology processes micro-volumes of fluid, typically tens to hundreds of micrometers in size, ranging from microliters to nanoliters or even attoliters. It enables precise manipulation of liquids at the micro- and nano-scale, allowing for most operations performed in conventional chemical and biological laboratories. Its advantages include small sample volume, high throughput, and ease of automation.

[0003] The core applications of microfluidic chips encompass microfluidic detection and analysis, serving as a key carrier for next-generation point-of-care testing (POCT) technology and an important form of in vitro diagnostics (IVD). Simultaneously, they can be used in various stages of sample preparation, reaction, separation, detection, cell culture, sorting, and lysis in the fields of biology and chemistry, finding wide application in clinical testing, food and environmental analysis, and virus screening. Microfluidic chips can also simulate the in vivo physiological environment, enabling the study of cell behavior under physiological or physiological-like conditions.

[0004] Leveraging their miniaturization and controllable microenvironment, microfluidic chips have been widely applied in cell migration and deformation research over the past 20 years. Existing research has developed this technology into a tool for functional phenotypic studies of cell migration and deformation-related diseases. For example, research groups have facilitated functional comparisons of cell motility characteristics under different disease states; experiments have shown that cell migration patterns and deformation characteristics under pathological conditions differ significantly from those in healthy states. For instance, research teams have previously developed a series of microfluidic chips for real-time monitoring of cell migration and achieved comparative studies of neutrophil chemotactic behavior in healthy individuals and sepsis patients under stimulation by stable chemokines (such as fMLP). Regarding cell deformability, Cytovale has launched the IntelliSep system based on microfluidic chips. This system assesses the biophysical properties of leukocytes through deformability cytology, aiding in the early diagnosis of sepsis and combining this assessment with clinical results to determine whether organ dysfunction will occur within three days of testing.

[0005] Despite significant advancements in existing technologies, key bottlenecks remain. First, there is a lack of physical-chemical coupling systems; current methods struggle to simultaneously simulate physiological-scale mechanical compression and stable chemical gradients, causing the microenvironment to deviate from physiological realities, leading to distortions in cell deformation rates and migration behavior. Second, single-cell behavior analysis capabilities are insufficient. Existing commercial software (such as MetaMorph®, Chemotaxis, and Migration Tool) primarily focuses on population-level migration speed and exponential analysis, failing to accurately quantify single-cell migration decision-making behavior or reveal the crucial link between cell deformation and migration. These shortcomings severely limit the efficiency of microfluidic platforms in single-cell-level migration and deformation research and their clinical translation. Summary of the Invention

[0006] One of the objectives of this invention is to provide a micropillar chip that solves the following technical problems: How to improve the efficiency of microfluidic platforms in single-cell hierarchical migration and deformation research and their clinical translation.

[0007] The second objective of this invention is to provide a preparation method for preparing the above-mentioned micropillar chip.

[0008] A third objective of this invention is to provide a method of use for employing the aforementioned micropillar chip.

[0009] The objective of this invention can be achieved through the following technical solutions: In a first aspect, the present invention discloses a micropillar chip, comprising a glass substrate and a PDMS chip disposed on one side of the glass substrate, characterized in that the PDMS chip is provided with cell migration and deformation channels, and the cell migration and deformation channels are provided with a plurality of micropillars. Also includes: A cell injection channel is connected downstream to a cell calibration channel, which is connected to a cell migration and deformation channel. The Christmas tree-shaped channel is connected downstream to the cell migration and deformation channel, and upstream to multiple sets of serpentine channels, each of which is connected upstream to a reagent injection channel. The waste liquid discharge channel is connected to the cell migration and deformation channel.

[0010] In a further embodiment of the present invention, the length × width × height dimensions of the cell migration and deformation channel are 1165 × 650 × 30 μm.

[0011] In a further embodiment of the present invention, the diameter × height of the micropillar is 25um × 30um, and the gap between two adjacent micropillars is 3-8um.

[0012] In a further embodiment of the present invention, the cell injection channel includes a cell injection port, a cell delivery channel, and a cell storage pool. The cell injection port is located on the surface of the PDMS chip, and the cell delivery channel and the cell storage pool are located inside the PDMS chip. The cell injection port is connected upstream of the cell delivery channel, and the cell storage pool is connected downstream of the cell delivery channel.

[0013] In a further embodiment of the present invention, the cell calibration channel is located inside the PDMS chip; the cell calibration channel is connected to the cell storage pool; and the height of the cell calibration channel is 5 μm.

[0014] In a further embodiment of the present invention, the reagent injection channel includes a reagent injection port and a reagent delivery channel. The reagent injection port is located on the surface of the PDMS chip, and the reagent delivery channel is located inside the PDMS chip. The reagent injection port is connected upstream of the reagent delivery channel, and the downstream of the reagent delivery channel is connected upstream of the corresponding serpentine channel.

[0015] In a further embodiment of the present invention, both the serpentine channel and the reagent injection channel are provided in two sets, and the two sets of reagent injection channels are respectively used to inject any one of culture medium, chemokine, drug or plasma reagent.

[0016] In a further embodiment of the present invention, the waste liquid discharge channel includes a waste liquid outlet and a waste liquid transport channel. The waste liquid outlet is located on the surface of the PDMS chip, and the waste liquid transport channel is located inside the PDMS chip. The waste liquid outlet is connected downstream of the waste liquid transport channel, and the waste liquid transport channel is connected upstream of the cell migration and deformation channel.

[0017] Secondly, the present invention discloses a method for preparing the micropillar chip as described above, comprising the following steps: Step 1: Obtain the micropillar chip prototype through modeling and design, and use photolithography to mold the PDMS chip in the micropillar chip prototype to obtain the mold of the PDMS chip. Step 2: After vacuum degassing, the PDMS solution is poured into a mold, then placed in an oven for curing. After cooling to room temperature, it is cut and demolded to obtain the PDMS chip. Step 3: Use anhydrous ethanol to ultrasonically clean the PDMS chip and the glass substrate, then use ultrapure water to ultrasonically clean them, and finally dry them in a nitrogen atmosphere for later use. Step 4: Perform plasma cleaning on the PDMS chip and the glass substrate; Step 5: Precisely align and bond the plasma-cleaned PDMS chip to the glass substrate to obtain the micropillar chip.

[0018] Thirdly, the present invention also discloses a method for using the micropillar chip as described above, comprising the following steps: S1. Prepare cell culture media and collagen solutions of different concentrations; S2. Resuspend the activated T cells or neutrophils that have just been isolated from the human body in cell culture medium and culture them at 37°C and 5% CO2. S3. Inject collagen solution into each channel of the PDMS chip to cover all channels; S4. Incubate the collagen solution for 0.5-1.5 hours, and then inject cell culture medium into each channel of the PDMS chip to cover all channels. S5. Place the PDMS chip in a microscope observation system at 37°C, adjust it to the area of ​​cell migration and deformation channels, and calibrate the focus. S6. Aspirate the cell culture medium, and then inject the T cells or neutrophils just isolated from the human body into the cell injection channel. The T cells or neutrophils just isolated from the human body enter the cell calibration channel and are neatly arranged. S7. For T cells, inject the cell culture medium into the corresponding reagent injection channels in equal volumes; for neutrophils, inject the chemokine solution and cell culture medium into the corresponding reagent injection channels in equal volumes; then, they enter the corresponding serpentine channels to achieve pressure equilibrium, and then enter the Christmas tree-shaped channels to merge and form a reagent solution with a linear concentration gradient in the longitudinal and transverse directions. S8 cells, T cells, or neutrophils newly isolated from the human body are stimulated by reagent solutions of different concentration gradients, which cause them to polarize and deform, enter cell migration and deformation channels, and exhibit migration direction selection and path decision-making behaviors, which are then tracked and analyzed.

[0019] In a further embodiment of the present invention, in S8, the tracking and analysis method is as follows: during the process of cell migration direction selection and path decision-making, 90 frames of images are collected at 20-second intervals. The images are manually tracked and analyzed by image processing software, and data processing is performed to obtain quantitative characterization data of migration speed, chemotaxis index, and deformation rate.

[0020] Preferably, the image processing software is ImageJ, and the data processing is performed using the Chemotaxis plugin, Matlab, and Origin software.

[0021] Preferably, based on the results of quantitative characterization, the differences in cell migration and deformation behavior and their phenotypic characteristics under different chemokine concentration gradients are analyzed, and the cell concentration response threshold is identified through data differences to screen effective reagent concentration ranges.

[0022] The present invention has at least the following beneficial effects: 1. The micropillar chip of the present invention includes a glass substrate and a PDMS chip disposed on one side of the glass substrate. The PDMS chip has a cell migration and deformation channel inside, and a plurality of micropillars are disposed in the cell migration and deformation channel. Through the coupling design of the micropillar channel formed by the plurality of micropillars and the stable chemical gradient, mechanical compression and chemotactic signals can be applied simultaneously on the basis of simulating the real microenvironment of cells, so as to realize highly biomimetic simulation of single cell migration and deformation behavior, breaking through the limitation of existing chips that can only simulate chemical gradient or mechanical compression alone. This micropillar chip features a finely structured, precisely controlled channel size, allowing only a single cell to pass through the gaps between the micropillars. The downstream of the Christmas tree-shaped channel connects to the cell migration and deformation channel, while the upstream connects to multiple sets of serpentine channels, each of which is connected to a reagent injection channel. Through the serpentine and Christmas tree-shaped channels, longitudinal and transverse gradient stimulation is formed, allowing observation of the dynamic decision-making, orientation selection, velocity changes, and deformability of single cells. The downstream of the cell injection channel connects to a cell calibration channel, which in turn connects to the cell migration and deformation channel. This ensures that all cells receive stimulation under uniform initial position and concentration conditions, effectively reducing experimental errors and improving data consistency and repeatability.

[0023] 2. The micropillar chip manufacturing process of this invention is mature and easy to replicate and promote: The chip adopts dual-layer SU-8 lithography and PDMS soft lithography technology. The preparation process is mature, the cost is low, and it is easy to standardize production, making it suitable for mass production and subsequent industrial applications.

[0024] 3. When using the micropillar chip of this invention, cell interaction and migration-deformation experiments can be performed, greatly improving screening efficiency and accuracy. Parameters such as cell concentration, flow rate, and temperature can be precisely controlled. The use of micro-volume liquids significantly reduces the amount of reagents and samples required, lowering experimental costs. Multiple cell migration and deformation tests can be completed simultaneously in a single experiment, supporting parallel processing. Low sample and reagent consumption makes it particularly suitable for analyzing valuable or hard-to-obtain clinical samples and immune cells. By setting up vascular endothelial stratification or multi-type cell injection ports, in vivo cell interaction processes can be simulated, making it suitable for research on inflammatory response mechanisms and evaluation of functional responses between immune cells and drugs. It has good application expansion and translational prospects. Attached Figure Description

[0025] The invention will now be further described with reference to the accompanying drawings.

[0026] Figure 1 This is a schematic diagram of the micropillar chip structure of Embodiment 1 of the present invention; Figure 2 This is a schematic diagram of the internal structure of the micropillar chip in Embodiment 1 of the present invention; Figure 3 yes Figure 2 A magnified schematic diagram of the internal structure of the PDMS chip in the micropillar chip; Figure 4 yes Figure 2 A magnified schematic diagram of a portion of the PDMS chip inside a micropillar chip; Figure 5 This is a schematic diagram of the micropillar structure in the micropillar chip of Embodiment 1 of the present invention; Figure 6 This is a diagram showing the concentration gradient test results of the micropillar chip prepared in Embodiment 2 of the present invention using Comsol simulation. Figure 7 These are observation images of T cells at 0 min and 30 min in Example 3 of this invention; Figure 8 This is a real-time motion diagram of T cells during migration and deformation in Embodiment 3 of the present invention; Figure 9 This is a real-time motion diagram of neutrophils during migration and deformation in Embodiment 4 of the present invention.

[0027] In the figure: 100, glass substrate; 200, PDMS chip; 2011, cell injection port; 2012, cell delivery channel; 2013, cell storage pool; 2021, waste liquid outlet; 2022, waste liquid delivery channel; 2031, chemokine injection port; 2032, chemokine delivery channel; 2041, cell culture medium injection port; 2042, cell culture medium delivery channel; 205, serpentine channel; 206, Christmas tree-shaped channel; 207, cell migration and deformation channel; 208, microcolumn; 209, cell calibration channel. Detailed implementation method.

[0028] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0029] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0030] Example 1

[0031] Please see Figure 1This embodiment discloses a micropillar chip, including a glass substrate 100 and a PDMS chip 200 disposed on one side of the glass substrate.

[0032] Please see Figure 2-3 The PDMS chip 200 contains a cell migration and deformation channel 207, which is rectangular in shape with dimensions of 1165 × 650 × 30 μm (length × width × height). Within the cell migration and deformation channel 207 are several micropillars 208, each with a diameter × height of 25 μm × 30 μm. (See [link to relevant documentation]). Figure 4-5 The gap between two adjacent micropillars 208 is 3-8 μm; it can effectively simulate the mechanical deformation and behavioral response of cells when they pass through the microstructure environment, and can be used to quantify the stress adaptation and migration path selection of cells in different microenvironments.

[0033] The PDMS chip 200 is also provided with a cell injection channel, which includes a cell injection port 2011, a cell delivery channel 2012, and a cell storage pool 2013. The cell injection port 2011 is located on the surface of the PDMS chip 200, while the cell delivery channel 2012 and the cell storage pool 2013 are located inside the PDMS chip 200. The cell injection port 2011 is connected upstream of the cell delivery channel 2012, and the cell storage pool 2013 is connected downstream of the cell delivery channel 2012.

[0034] The PDMS chip 200 is also equipped with a cell calibration channel 209, which is located inside the PDMS chip 200. One side of the cell calibration channel 209 is connected to the cell storage pool 2013, and the other side is connected to the cell migration and deformation channel 207. The height of the cell calibration channel 209 is 5 μm. When the cell suspension enters from the cell injection channel, since the cell diameter range is 10-15 μm and the height of the cell calibration channel 209 is 5 μm, it helps to restrict cells of different sizes to a uniform starting position, thereby ensuring that their migration behavior after receiving chemotactic signals is comparable and consistent.

[0035] The PDMS chip 200 also has a Christmas tree-shaped channel 206, which is connected downstream to the cell migration and deformation channel 207 and upstream to two sets of serpentine channels 205. Each set of serpentine channels 205 is connected upstream to a reagent injection channel. The two sets of reagent injection channels are used to inject any one of culture medium, chemokines, drugs or plasma reagents, respectively. The serpentine channels 205 and the Christmas tree-shaped channel 206 together form a gradient network, which can promote the formation of a stable longitudinal and transverse concentration gradient of reagents in the cell migration and deformation channel 207.

[0036] In this embodiment, the two sets of reagent channels are a chemokine channel and a cell culture medium channel. The chemokine channel includes a chemokine injection port 2031 and a chemokine transport channel 2032. The chemokine injection port 2031 is located on the surface of the PDMS chip 200, and the chemokine transport channel 2032 is located inside the PDMS chip 200. The chemokine injection port 2031 is connected upstream to the chemokine transport channel 2032, and the downstream of the chemokine transport channel 2032 is connected upstream to the corresponding serpentine channel 205. The cell culture medium channel includes a cell culture medium injection port 2041 and a cell culture medium transport channel 2042. The cell culture medium injection port 2041 is located on the surface of the PDMS chip 200, and the cell culture medium transport channel 2042 is located inside the PDMS chip 200. The cell culture medium injection port 2041 is connected upstream to the cell culture medium transport channel 2042, and the downstream of the cell culture medium transport channel 2042 is connected upstream to the corresponding serpentine channel 205.

[0037] The PDMS chip 200 is also provided with a waste liquid discharge channel, which includes a waste liquid outlet 2021 and a waste liquid transport channel 2022. The waste liquid outlet 2021 is located on the surface of the PDMS chip 200, and the waste liquid transport channel 2022 is located inside the PDMS chip 200. The waste liquid outlet 2021 is connected to the downstream of the waste liquid transport channel 2022, and the upstream of the waste liquid transport channel 2022 is connected to the cell migration and deformation channel 207.

[0038] Example 2

[0039] This embodiment discloses a method for preparing the micropillar chip of Embodiment 1, including the following steps: Step 1: The micropillar chip prototype is obtained by modeling and designing with SolidWorks software, and the PDMS chip 200 in the micropillar chip prototype is molded using photolithography technology to obtain the mold of PDMS chip 200. Step 2: The PDMS chip 200 body needs to be made of high-purity polydimethylsiloxane (PDMS, which is prepared by mixing the base adhesive and curing agent at a mass ratio of 10:1). After vacuum degassing, it is poured into a mold with a preset micro-pillar structure, placed in a 75°C oven for curing for 2 hours, and then cut and demolded after cooling to room temperature to obtain the PDMS chip 200 with micro-pillar array channels. Step 3: Select borosilicate glass (0.5mm thick, size matching the PDMS chip body) for the glass substrate 100. After ultrasonic cleaning with anhydrous ethanol, ultrasonic cleaning with ultrapure water is performed. Finally, place it under a nitrogen dryer to dry for later use.

[0040] Step 4: Place the processed PDMS chip 200 (micro-pillar channel side up) and glass substrate 100 (clean side up) into the sample chamber of the plasma cleaner, close the sample chamber door, start the equipment, and after reaching the preset power and time, stop plasma generation and slowly release the gas to atmospheric pressure.

[0041] Step 5: Immediately remove the PDMS chip 200 and the glass substrate 100. In a sterile laminar flow hood, using the edge positioning marks of the PDMS chip 200 as a reference (alignment lines can be marked on the edges of the PDMS and glass substrate beforehand with a sterile marker), precisely align the micropillar channel surface of the PDMS chip 200 with the clean surface of the glass substrate 100. Gently press the edges of the PDMS chip 200 with clean tweezers to ensure initial bonding without gaps. At this point, the bonding strength is sufficient for short-term liquid injection, and no pressure from weights or oven drying is required. After bonding is complete, the total time from removal from plasma cleaning to initial bonding should be controlled within 30 seconds to prevent the hydrophilicity of the PDMS chip 200 surface from decreasing over time, thus obtaining the micropillar chip.

[0042] It is important to note that before using the micropillar chip, collagen solution must be injected into the chip to cover all channels and incubated for 60 minutes to promote cell adhesion, rolling, and crawling on the glass surface; then, cell culture medium should be injected to cover all channels and incubated for another 30 minutes. One micropillar chip is used for each experiment.

[0043] Specifically, for the preparation of the collagen solution, if performing T cell testing, type I collagen was selected at a target concentration of 5 μg / mL; if performing neutrophil testing, fibronectin was selected at a target concentration of 2.5 μg / mL. Both solutions were prepared by dilution with PBS and stored at 4°C after preparation. For the preparation of the cell culture medium, RPMI-1640 medium was used as the basal medium, with 5% (or 10%) fetal bovine serum (FBS) and 1% penicillin / streptomycin mixture added to prepare the basic cell culture solution.

[0044] The specific operation process is as follows: After step five, immediately transfer the chip to a sterile operating area without additional settling. Directly proceed with the Collagen solution injection process. The Collagen solution's infiltration and retention within the microcolumn channels further maintains the channel's hydrophilicity and simultaneously constructs a biocompatible interface for subsequent cell experiments. This prevents the PDMS surface from becoming hydrophobic again due to oven settling, which could affect the subsequent solution spreading effect. After settling for 30 minutes, in a sterile laminar flow hood, remove the sealing film and use a 200μL sterile pipette. Gently align the pipette tip with the chip's outlet and slowly aspirate the Collagen solution from the channel to form a Collagen gel coating. After aspiration, gently wipe away any remaining solution around the outlet with sterile filter paper to avoid contamination. Then, use a sterile pipette to draw up the preheated cell culture solution and slowly inject it into the channel through the inlet, ensuring that the cell culture solution completely infiltrates the Collagen gel coating and that there are no air bubbles within the channel. After injection, the inlet and outlet ports are resealed with sterile sealing film, and the chip is placed back into a 37°C, 5% CO2 incubator and left to stand for 1 hour to allow the cell culture solution and Collagen gel coating to fully equilibrate, laying the foundation for subsequent cell seeding and experiments. After standing, the micropillar structure, Collagen coating and solution state in the channel are observed using an inverted phase contrast microscope. Once it is confirmed that there is no leakage, no air bubbles and the coating is intact, it can be used for cell interaction, single-cell migration and deformation research experiments.

[0045] Please see Figure 6 After preparation, the micropillar chip is tested. The testing steps are as follows: Step 1: Prepare fluoresein-FITC fluorescent solution and RPMI-1640 solution containing FBS (FBS molar concentration of 5%). Step 2: Place the prepared micropillar chip into the microscope system, adjust the focus and set the fluorescence observation mode and parameters; Step 3: Using an injection pump, simultaneously inject 100 μL of fluoresein-FITC fluorescent solution and RPMI 1640 solution from Step 1 into chemokine injection port 2031 and cell culture medium injection port 2041, respectively. Step 4: Observe the fluorescence intensity in cell migration and deformation channel 207 in real time, and set the microscope imaging time parameters to continuously image for 30 minutes; Step 5: Observe the changes in fluorescence images to determine the duration of the concentration gradient and to determine the stable gradient concentration that can be formed in cell migration and deformation channel 207.

[0046] Example 3

[0047] This embodiment discloses the method of using the micropillar chip of Example 1 for analyzing the spontaneous migration-deformation motion characteristics of T cells; the micropillar chip of this embodiment is prepared according to Example 2, including the following steps: S1. Prepare RPMI-1640 solution (T cell culture medium) with 10% FBS and Collagen solution with a concentration of 5 μg / mL. S2. After isolating T cells from the blood, place them in an incubator, add CD3 and CD28 antibodies to activate the T cells for 48 hours, then change the medium and passage every 3 days, adding IL-2 to maintain the activated state; after culturing to the 3rd generation, observe the T cell culture status, take out a portion of the cell suspension and resuspend it in the RPMI-1640 solution prepared in S1 to form a cell suspension, and control the T cell concentration in the T cell suspension at 1 × 10⁻⁶. 6 cell / mL; cultured at 37℃ in a 5% CO2 incubator; S3. Inject the Collagen solution prepared in S1 into each channel of the PDMS chip 200 to cover all channels; S4. After culturing the Collagen solution for 1 hour, aspirate it and then inject RPMI-1640 solution into each channel of the PDMS chip 200 to cover all channels and maintain for 30 minutes. S5. Place the PDMS chip 200 in a microscope observation system at 37°C. Set the microscope to 37°C, adjust it to the area of ​​the cell migration and deformation channel 207, and calibrate the focus. S6. Aspirate the RPMI-1640 solution, then inject S2 culture solution at a concentration of 1×10⁻⁶ into cell injection port 2011. 6 A T-cell suspension of cells / mL was prepared, with T cells flowing from cell delivery channel 2012 into cell storage pool 2013; T cells were observed under a microscope until they were neatly arranged at the edge of cell calibration channel 209. S7. RPMI-1640 solution is injected into the chemokine inlet 2031 and the cell culture medium inlet 2041 in equal volumes using an injection pump. Then, it enters the corresponding serpentine channel 205 through the chemokine delivery channel 2032 and the cell culture medium delivery channel 2042 to achieve pressure balance. It then enters the Christmas tree-shaped channel 206 to merge and form a reagent solution with a linear concentration gradient in the longitudinal and transverse directions. The reagent solution with a linear concentration gradient in the longitudinal and transverse directions enters the cell migration and deformation channel 207. S8, please refer to Figure 7-8T cells, stimulated by reagent solutions of different concentration gradients, undergo polarization and deformation, entering cell migration and deformation channel 207. They exhibit migration direction selection and path decision-making behaviors, which are then tracked and analyzed. During this migration direction selection and path decision-making process, 90 images were acquired at 20-second intervals using a microscope. Manual tracking and analysis were performed using ImageJ image processing software, and data processing was conducted using the Chemotaxis plugin, Matlab, and Origin software to obtain quantitative characterization data of migration speed, chemotactic index, and deformation rate. Based on the results of the quantitative characterization data, the differences in cell migration and deformation behaviors and their phenotypic characteristics under different chemokine concentration gradients were analyzed. Cell concentration response thresholds were identified through data differences, and effective reagent concentration ranges were screened.

[0048] Example 4

[0049] This embodiment discloses the method of using the micropillar chip of Example 1 for analyzing the spontaneous migration-deformation movement characteristics of neutrophils; the micropillar chip of this embodiment is prepared according to Example 2, including the following steps: S1. Prepare 100 nM fMLP solution (chemokine), 5% FBS RPMI-1640 solution (neutrophil culture medium), and 2.5 μg / mL Fibronectin solution (collagen solution). S2. Neutrophils were isolated from the blood and placed in an incubator. CD3 and CD28 antibodies were added to activate the neutrophils for 48 hours. The medium was changed and passaged every 3 days, and IL-2 was added to maintain the activated state. After culturing to the 3rd generation, the neutrophil culture status was observed. A portion of the cell suspension was taken out and resuspended in the RPMI-1640 solution prepared in S1 to form a cell suspension. The neutrophil concentration in the neutrophil suspension was controlled at 1 × 10⁻⁶. 6 cell / mL; cultured at 37℃ in a 5% CO2 incubator; S3. Inject the Fibronectin solution prepared in S1 into each channel of the PDMS chip 200 to cover all channels; S4. After culturing the Fibronectin solution for 1 hour, aspirate it and then inject RPMI-1640 solution into each channel of the PDMS chip 200 to cover all channels and maintain for 30 minutes. S5. Place the PDMS chip 200 in a microscope observation system at 37°C. Set the microscope to 37°C, adjust it to the area of ​​the cell migration and deformation channel 207, and calibrate the focus. S6. Aspirate the RPMI-1640 solution, then inject S2 culture solution at a concentration of 1×10⁻⁶ into cell injection port 2011. 6A neutrophil suspension of cells / mL was prepared, with neutrophils flowing from cell delivery channel 2012 into cell storage pool 2013; neutrophils were observed under a microscope until they were neatly arranged at the edge of cell calibration channel 209. S7. Simultaneously inject fMLP solution and RPMI-1640 solution in equal volumes into chemokine injection port 2031 and cell culture medium injection port 2041 via an injection pump. Then, they enter the corresponding serpentine channel 205 through chemokine delivery channel 2032 and cell culture medium delivery channel 2042 respectively to achieve pressure balance. They then enter the Christmas tree-shaped channel 206 to merge and form a reagent solution with a linear concentration gradient in the longitudinal and transverse directions. The reagent solution with a linear concentration gradient in the longitudinal and transverse directions enters the cell migration and deformation channel 207. S8, please refer to Figure 9 Neutrophils, stimulated by reagent solutions of different concentration gradients, undergo polarization and deformation, entering cell migration and deformation channel 207. They exhibit migration direction selection and path decision-making behaviors, which are then tracked and analyzed. During this migration direction selection and path decision-making process, 90 images were acquired at 20-second intervals using a microscope. Manual tracking and analysis were performed using ImageJ image processing software, and data processing was conducted using the Chemo neutrophil axis plugin, Ma neutrophil lab, and Origin software to obtain quantitative characterization data of migration speed, chemotactic index, and deformation rate. Based on the results of the quantitative characterization data, the differences in cell migration and deformation behaviors and their phenotypic characteristics under different chemokine concentration gradients were analyzed. Cell concentration response thresholds were identified through data differences, and effective reagent concentration ranges were screened.

[0050] Example 5

[0051] This embodiment discloses the method of using the micropillar chip of Example 1 for analyzing the motility characteristics of the interaction between T cells and endothelial cells; the micropillar chip of this embodiment is prepared according to Example 2, including the following steps: S1. Prepare RPMI-1640 solution (T cell culture medium) with 10% FBS, Collagen solution with a concentration of 5 μg / mL, and RPMI-1640 culture medium (endothelial cell culture medium) with 10% FBS and 1% penicillin. S2. After isolating T cells from the blood, place them in an incubator, add CD3 and CD28 antibodies to activate the T cells for 48 hours, then change the medium and passage every 3 days, adding IL-2 to maintain the activated state; after culturing to the 3rd generation, observe the T cell culture status, take out a portion of the cell suspension and resuspend it in the RPMI-1640 solution prepared in S1 to form a cell suspension, and control the T cell concentration in the T cell suspension at 1 × 10⁻⁶. 6cell / mL; cultured at 37℃ in a 5% CO2 incubator; with a culture concentration of 1×10⁻⁶ cells / mL. 5 Endothelial cell suspension at cell / mL; S3. Inject the Collagen solution prepared in S1 into each channel of the PDMS chip 200 to cover all channels; S4. After culturing the Collagen solution for 1 hour, aspirate it and then inject endothelial cell culture medium into each channel of the PDMS chip 200 to cover all channels and maintain for 30 minutes. S5. Place the PDMS chip 200 in a microscope observation system at 37°C. Set the microscope to 37°C, adjust it to the area of ​​the cell migration and deformation channel 207, and calibrate the focus. S6. Aspirate the endothelial cell culture medium, and then inject S2 culture medium at a concentration of 1×10⁻⁶ into cell injection port 2011. 5 Endothelial cell suspension at cell / mL; Endothelial cells flow from cell delivery channel 2012 into cell storage pool 2013; endothelial cells are observed under a microscope until they are neatly arranged at the edge of cell calibration channel 209, and then endothelial cell culture medium is injected into each injection port simultaneously through a syringe pump, and cultured in a humidified incubator at 37 °C containing 5% CO2 for 3 hours until the endothelial cells are completely spread out; S7, then inject S2 culture at a concentration of 1×10⁻⁶ into cell injection port 2011. 6 T cell suspension at cell / mL is introduced, and T cells flow from cell delivery channel 2012 into cell storage pool 2013. T cells are observed under a microscope until they are neatly arranged at the edge of cell calibration channel 209. Then, endothelial cell culture medium and T cell culture medium are simultaneously injected into chemokine injection port 2031 and cell culture medium injection port 2041 in equal volumes using an injection pump. They then enter the corresponding serpentine channel 205 through chemokine delivery channel 2032 and cell culture medium delivery channel 2042 to achieve pressure balance, and then enter the Christmas tree-shaped channel 206 to merge and form a reagent solution with a linear concentration gradient in the longitudinal and transverse directions. The reagent solution with a linear concentration gradient in the longitudinal and transverse directions enters the cell migration and deformation channel 207. S8, please refer to Figure 7-8T cells, stimulated by reagent solutions of different concentration gradients, undergo polarization and deformation, entering cell migration and deformation channel 207. They exhibit migration direction selection and path decision-making behaviors, which are then tracked and analyzed. During this migration direction selection and path decision-making process, 90 images were acquired at 20-second intervals using a microscope. Manual tracking and analysis were performed using ImageJ image processing software, and data processing was conducted using the Chemotaxis plugin, Matlab, and Origin software to obtain quantitative characterization data of migration speed, chemotactic index, and deformation rate. Based on the results of the quantitative characterization data, the differences in cell migration and deformation behaviors and their phenotypic characteristics under different chemokine concentration gradients were analyzed. Cell concentration response thresholds were identified through data differences, and effective reagent concentration ranges were screened.

[0052] Example 6

[0053] This embodiment discloses the method of using the micropillar chip of Example 1 for analyzing the motility characteristics of the interaction between neutrophils and endothelial cells; the micropillar chip of this embodiment is prepared according to Example 2, including the following steps: S1. Prepare RPMI-1640 solution (neutrophil culture medium) with 5% FBS, Collagen solution (collagen solution) with a concentration of 5 μg / mL, and RPMI-1640 culture medium (endothelial cell culture medium) with 10% FBS and 1% penicillin. S2. Neutrophils were isolated from the blood and placed in an incubator. CD3 and CD28 antibodies were added to activate the neutrophils for 48 hours. The medium was changed and passaged every 3 days, and IL-2 was added to maintain the activated state. After culturing to the 3rd generation, the neutrophil culture status was observed. A portion of the cell suspension was taken out and resuspended in the RPMI-1640 solution prepared in S1 to form a cell suspension. The neutrophil concentration in the neutrophil suspension was controlled at 1 × 10⁻⁶. 6 cell / mL; cultured at 37℃ in a 5% CO2 incubator; with a culture concentration of 1×10⁻⁶ cells / mL. 5 Endothelial cell suspension at cell / mL; S3. Inject the Collagen solution prepared in S1 into each channel of the PDMS chip 200 to cover all channels; S4. After culturing the Collagen solution for 1 hour, aspirate it and then inject endothelial cell culture medium into each channel of the PDMS chip 200 to cover all channels and maintain for 30 minutes. S5. Place the PDMS chip 200 in a microscope observation system at 37°C. Set the microscope to 37°C, adjust it to the area of ​​the cell migration and deformation channel 207, and calibrate the focus. S6. Aspirate the endothelial cell culture medium, and then inject S2 culture medium at a concentration of 1×10⁻⁶ into cell injection port 2011. 5 Endothelial cell suspension at cell / mL; Endothelial cells flow from cell delivery channel 2012 into cell storage pool 2013; endothelial cells are observed under a microscope until they are neatly arranged at the edge of cell calibration channel 209, and then endothelial cell culture medium is injected into each injection port simultaneously through a syringe pump, and cultured in a humidified incubator at 37 °C containing 5% CO2 for 3 hours until the endothelial cells are completely spread out; S7, then inject S2 culture at a concentration of 1×10⁻⁶ into cell injection port 2011. 6 Neutrophil suspension at cell / mL is introduced, with neutrophils flowing from cell delivery channel 2012 into cell storage pool 2013; neutrophils are observed under a microscope until they are neatly arranged at the edge of cell calibration channel 209; then, endothelial cell culture medium and neutrophil culture medium are simultaneously injected in equal volumes into chemokine inlet 2031 and cell culture medium inlet 2041 via an injection pump, respectively. They then enter the corresponding serpentine channel 205 through chemokine delivery channel 2032 and cell culture medium delivery channel 2042 to achieve pressure balance, and then enter the Christmas tree-shaped channel 206 to merge and form a reagent solution with a linear concentration gradient in the longitudinal and transverse directions. The reagent solution with a linear concentration gradient in the longitudinal and transverse directions enters the cell migration and deformation channel 207; S8, please refer to Figure 7-8 Neutrophils, stimulated by reagent solutions of different concentration gradients, undergo polarization and deformation, entering cell migration and deformation channel 207. They exhibit migration direction selection and path decision-making behaviors, which are then tracked and analyzed. During the cell migration direction selection and path decision-making process, 90 images were acquired at 20-second intervals. Manual tracking and analysis were performed using ImageJ image processing software, and data processing was conducted using the Chemotaxis plugin, Matlab, and Origin software to obtain quantitative characterization data of migration speed, chemotactic index, and deformation rate. Based on the results of the quantitative characterization data, the differences in cell migration and deformation behaviors and their phenotypic characteristics under different chemokine concentration gradients were analyzed. Cell concentration response thresholds were identified through data differences, and effective reagent concentration ranges were screened.

[0054] In the description of this invention, it should be understood that the terms "upper," "lower," "left," and "right," etc., indicating orientation or positional relationships based on the orientation or positional relationships shown in the accompanying drawings, are only for the convenience of describing the invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation or specific orientational structure and operation. Therefore, they should not be construed as limitations on the invention. Furthermore, "first" and "second" are only for descriptive purposes and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Therefore, a feature defined with "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of this invention, unless otherwise stated, "multiple" means two or more.

[0055] In the description of this invention, it should be noted that, unless otherwise explicitly specified and limited, the terms "installation," "connection," "linking," etc., should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral connection; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; and they can refer to the internal communication between two components. Those skilled in the art can understand the specific meaning of the above terms in this invention based on the specific circumstances.

[0056] The foregoing has described several embodiments of the present invention in detail, but these descriptions are merely preferred embodiments and should not be construed as limiting the scope of the invention. All equivalent variations and modifications made within the scope of the claims of this invention should still fall within the patent coverage of this invention.

Claims

1. A micropillar chip, comprising a glass substrate (100) and a PDMS chip (200) disposed on one side of the glass substrate, characterized in that, The PDMS chip (200) has a cell migration and deformation channel (207) inside, and the cell migration and deformation channel (207) has a number of micropillars (208). Also includes: A cell injection channel is connected downstream to a cell calibration channel (209), which is connected to a cell migration and deformation channel (207); The Christmas tree-shaped channel (206) is connected downstream to the cell migration and deformation channel (207) and upstream to multiple sets of serpentine channels (205), each set of serpentine channels (205) being connected upstream to a reagent injection channel; The waste liquid discharge channel is connected to the cell migration and deformation channel (207).

2. The micropillar chip according to claim 1, characterized in that, The length × width × height of the cell migration and deformation channel (207) is 1165 × 650 × 30 μm.

3. The micropillar chip according to claim 2, characterized in that, The micropillar (208) has a diameter × height of 25um × 30um, and the gap between two adjacent micropillars (208) is 3-8um.

4. The micropillar chip according to claim 1, characterized in that, The cell injection channel includes a cell injection port (2011), a cell delivery channel (2012), and a cell storage pool (2013). The cell injection port (2011) is located on the surface of the PDMS chip (200), and the cell delivery channel (2012) and the cell storage pool (2013) are located inside the PDMS chip (200). The cell injection port (2011) is connected upstream of the cell delivery channel (2012), and the cell storage pool (2013) is connected downstream of the cell delivery channel (2012).

5. The micropillar chip according to claim 1, characterized in that, The cell calibration channel (209) is located inside the PDMS chip (200); the cell calibration channel (209) is connected to the cell storage pool (2013); the height of the cell calibration channel (209) is 5 μm.

6. The micropillar chip according to claim 1, characterized in that, The reagent injection channel includes a reagent injection port and a reagent delivery channel. The reagent injection port is located on the surface of the PDMS chip (200), and the reagent delivery channel is located inside the PDMS chip (200). The reagent injection port is connected to the upstream of the reagent delivery channel, and the downstream of the reagent delivery channel is connected to the upstream of the corresponding serpentine channel (205).

7. The micropillar chip according to claim 1, characterized in that, Both the serpentine channel (205) and the reagent injection channel are provided in two sets, and the two sets of reagent injection channels are used to inject any one of culture medium, chemokine, drug or plasma reagent.

8. The micropillar chip according to claim 1, characterized in that, The waste liquid discharge channel includes a waste liquid outlet (2021) and a waste liquid transport channel (2022). The waste liquid outlet (2021) is located on the surface of the PDMS chip (200), and the waste liquid transport channel (2022) is located inside the PDMS chip (200). The waste liquid outlet (2021) is connected downstream of the waste liquid transport channel (2022), and the waste liquid transport channel (2022) is connected upstream of the cell migration and deformation channel (207).

9. A preparation method for preparing the micropillar chip as described in any one of claims 1-8, characterized in that, Includes the following steps: Step 1: Obtain the micropillar chip prototype through modeling and design, and use photolithography to mold the PDMS chip (200) in the micropillar chip prototype to obtain the mold of the PDMS chip (200); Step 2: After vacuum degassing, the PDMS solution is poured into a mold, then placed in an oven for curing. After cooling to room temperature, it is cut and demolded to obtain a PDMS chip (200). Step 3: Use anhydrous ethanol to ultrasonically clean the PDMS chip (200) and the glass substrate (100), then use ultrapure water to ultrasonically clean them, and then dry them in a nitrogen atmosphere for later use. Step 4: Perform plasma cleaning on the PDMS chip (200) and the glass substrate (100); Step 5: Align and bond the plasma-cleaned PDMS chip (200) with the glass substrate (100) to obtain the micropillar chip.

10. A method of using the micropillar chip as described in any one of claims 1-8, characterized in that, Includes the following steps: S1. Prepare cell culture media and collagen solutions of different concentrations; S2. Resuspend the activated T cells or neutrophils that have just been isolated from the human body in cell culture medium and culture them at 37°C and 5% CO2. S3. Inject collagen solution into each channel of the PDMS chip (200) to cover all channels; S4. Incubate the collagen solution for 0.5-1.5 hours, and then inject cell culture medium into each channel of the PDMS chip (200) to cover all channels; S5. Place the PDMS chip (200) in a microscope observation system at 37°C, adjust it to the area of ​​the cell migration and deformation channel (207), and calibrate the focus. S6. Aspirate the cell culture medium and then inject the T cells or neutrophils just isolated from the human body into the cell injection channel. The T cells or neutrophils just isolated from the human body enter the cell calibration channel (209) and are neatly arranged. S7. For T cells, inject the cell culture medium into the corresponding reagent injection channels in equal volumes; For neutrophils, chemokine solution and cell culture medium were injected into the corresponding reagent injection channels in equal volumes. Then they enter the corresponding serpentine channels (205) to achieve pressure balance, and then enter the Christmas tree-shaped channel (206) to merge and form a reagent solution with a linear concentration gradient in the longitudinal and transverse directions; S8 cells, T cells, or neutrophils just isolated from the human body are stimulated by reagent solutions of different concentration gradients, which cause polarization and deformation. They enter the cell migration and deformation channel (207), exhibit migration direction selection and path decision-making behavior, and are tracked and analyzed.

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