Microstructure spiral inertial microfluidic chip for cell deformation ability sorting and application thereof

Abstract

The present application relates to the technical field of microfluidic chip, and specifically discloses a microstructure spiral inertia microfluidic chip for cell deformation ability sorting and application thereof, the chip comprises a spiral flow channel, an inlet is arranged at the upstream end, a first outlet and a second outlet are arranged at the downstream end, and micro-pillars are arranged on the inner side wall of the spiral flow channel towards the curvature center at equal intervals. The present application strengthens the Dean secondary flow through the micro-pillar structure, and in a wide flow rate range, cells with different deformation abilities are respectively focused on the near inner wall and far inner wall regions of the flow channel based on the force balance difference, and high-throughput sorting without barriers is realized through the corresponding outlets, the sorting flux is more than 2 million cells / min, the migration and invasion ability of the soft cancer cell subpopulation obtained by sorting is 2-3 times that of hard cells, high-speed imaging can be matched to realize online quality control, and technical support is provided for tumor mechanical phenotype research.

Description

Technical Field

[0001] This invention relates to the field of microfluidic chip technology, specifically to a microstructured helical inertial microfluidic chip for sorting cell deformability and its application. Background Technology

[0002] The mechanical deformability (cell stiffness) of cancer cells is a key indicator reflecting their malignancy and metastatic potential. Studies have shown that cancer cells with higher deformability (i.e., softer cells) typically exhibit stronger migration and invasion capabilities, which is closely related to changes in the expression of epithelial-mesenchymal transition (EMT)-related proteins. Therefore, isolating cancer cell subpopulations with different deformability is of great significance for elucidating tumor metastasis mechanisms and achieving personalized diagnosis.

[0003] Microfluidics provides a powerful tool for measurement and cell deformability analysis. Existing microfluidic cell sorting patents primarily rely on physical parameters such as cell size and electrical properties for sorting. However, these methods are typically highly sensitive to perfusion flow rate and cell size, have narrow operating windows, and are prone to causing mechanical damage to cells and channel blockage, making it difficult to achieve high-throughput and stable operation. Therefore, existing technologies cannot directly adapt to the sorting requirements based on cell deformability, and a sorting scheme that is easy to prepare, high-throughput, and with low cell damage is still lacking.

[0004] In the field of helical inertial microfluidics, ordinary helical channels can only achieve position observation, but the secondary flow stability is poor and the flux is low, making it impossible to achieve continuous separation of cells with different stiffness. Summary of the Invention

[0005] To develop a high-throughput, low-damage device capable of stably sorting cancer cells with varying deformability, this invention provides a microstructured helical inertial microfluidic chip for cell deformability sorting and its application. The microstructured helical inertial microfluidic chip provided by this invention stabilizes and accelerates the Dean secondary flow by introducing ordered micropillar structures into the helical microchannels, expanding the operational window for particle focusing and separation. This enables high-throughput, low-damage sorting of cancer cells based on their deformability and can be combined with real-time image analysis to study the correlation between cell stiffness, protein expression, and metastatic ability.

[0006] This invention provides a microstructured helical inertial microfluidic chip for sorting cell deformability, comprising a helical channel, wherein a plurality of micropillars are equidistantly arranged on the inner sidewall of the helical channel facing the center of curvature along the length of the helical channel; an inlet is provided at the upstream end of the helical channel, and a first outlet and a second outlet are provided at the downstream end. The micro-column is integrally formed by connecting a lower rectangular section and an upper semi-circular arch section. The straight bottom edge of the semi-circular arch section is completely fitted with the upper surface of the rectangular section, and the overall top surface is a smooth arc raised within the spiral flow channel.

[0007] This invention incorporates ordered micropillars within a helical flow channel to enhance Dean's secondary flow and create a side-biased velocity distribution on the inner wall, enabling cells to achieve stable single-line focusing across a wide velocity range. Utilizing the balance between shear gradient lift, wall-induced lift, Dean's drag, and deformability-induced lift, cells with different deformability experience varying forces across the cross-section: hard cells, due to lower deformability lift, are balanced near the inner wall; soft cells, with higher deformability lift, work in conjunction with wall lift to push towards the outer wall, occupying different lateral positions. Spatial separation is achieved through downstream extension, enabling high-throughput sorting based on cell stiffness without the need for barriers.

[0008] Furthermore, the spiral channel has a planar single spiral configuration with 4 to 6 spiral turns and a spiral spacing of 200 μm to 400 μm; the cross-sectional height is 40 μm to 60 μm, the width is 800 μm to 1000 μm, and the initial radius of curvature is 2 mm to 4 mm.

[0009] Furthermore, the central angle between adjacent micropillars and the center point of the spiral channel is π / 8 to π / 4, and a total of 10 to 15 micropillars are set per cycle.

[0010] Furthermore, the length of the cuboid segment is 300 μm to 400 μm, the width is 150 μm to 250 μm, and the height is 40 μm to 60 μm; the diameter of the semi-circular arch segment is 150 μm to 250 μm, and the height is 40 μm to 60 μm.

[0011] Furthermore, the cross-sectional width of the first outlet is 100 μm to 200 μm and the cross-sectional height is 40 μm to 60 μm; the cross-sectional width of the second outlet is 200 μm to 400 μm and the cross-sectional height is 40 μm to 60 μm.

[0012] This invention also provides the application of the aforementioned microstructured helical inertial microfluidic chip in sorting cells of different hardness.

[0013] Furthermore, the specific steps include: The cells to be sorted were resuspended in PBS buffer containing bovine serum albumin and the cell concentration was adjusted to 1.0 × 10⁶ cells / day. 6 cells / mL ~1.5×10 6 cells / mL; The cell suspension was injected at a constant rate of 1.2 mL / min to 2.0 mL / min into the inlet of the spiral channel using a syringe pump. Cells migrate laterally within the spiral channel due to the Dean secondary flow enhanced by micropillars. Hard cells with weak deformability focus on the channel region near the inner wall and are collected from the first outlet; soft cells with strong deformability focus on the channel region away from the inner wall and are collected from the second outlet, thus completing the sorting of cells with different hardness.

[0014] Furthermore, it also includes a real-time cell morphology and phenotypic analysis step: a high-speed imaging system is built in the outlet area of ​​the spiral flow channel to acquire real-time bright-field images of the flowing cells, automatically identify individual cells and extract cell area, perimeter, and roundness morphological parameters, and quantify cell deformability through the deformability index to achieve online quality control of the sorting process.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows: The chip of this invention features an ordered micropillar structure within a helical flow channel. This structure enhances the Dean secondary flow and creates a stable, skewed velocity distribution on the inner wall of the channel cross-section, enabling cells with the same deformability to achieve single-line focusing within a wide flow rate range of 1.2 mL / min to 2.0 mL / min. Cells with different deformability migrate to different lateral positions within the channel. In this invention, the lateral position of a cell within the helical microchannel cross-section is determined by the following force balance: FLS + FD = FLW + FDD, where FLS is the shear gradient lift, related to cell diameter and local shear rate; FLW is the wall-induced lift, related to the distance between the cell and the wall; FDD is the Dean drag, related to the Dean number and cell size; and FD is the deformability-induced lift, positively correlated with cell deformability. For cells with weak deformability (hard cells), F~D~ is smaller, and the cell reaches equilibrium near the inner wall under a smaller resultant force. For cells with strong deformability (soft cells), F~D~ is significantly larger, and works synergistically with F~LW~ to push the cell towards the outer wall until a new equilibrium is reached near the outer wall. Thus, soft and hard cells occupy different lateral equilibrium positions within the channel cross-section, which, after extending downstream, translate into spatial separation at the outlet, thereby achieving barrier-free high-throughput sorting based on cell stiffness.

[0016] Within the aforementioned flow rate range, this invention achieves ultra-high throughput sorting exceeding 2,000,000 cells / minute (cell suspension concentration 1.2 × 10⁻⁶). 6 The sample volume is 1.7 mL / min (cells / mL, injection flow rate 1.7 mL / min). The channel is not prone to clogging, the sorting process causes minimal mechanical damage to cells, and the collected cells have good viability and can be directly used for subsequent culture and functional analysis.

[0017] The low Young's modulus soft cancer cell subpopulation obtained by the present invention has a migration and invasion ability that is 2 to 3 times that of the hard cell subpopulation. The soft cells also exhibit upregulated expression of vimentin and downregulated expression of E-cadherin, accompanied by mesenchymal-like morphology and cytoskeleton remodeling, which verifies the negative correlation between cell stiffness and tumor metastatic potential.

[0018] This invention can be used in conjunction with image-based cell parameter phenotypic analysis technology to obtain morphological features related to cell mechanics without additional labeling, providing reliable technical support for liquid biopsy based on cell mechanical properties, personalized diagnosis, and high-throughput drug screening. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 This is a structural diagram of the chip of the present invention.

[0021] Figure 2 This invention demonstrates the chip's ability to sort cells with different deformability. In the figure, A shows the movement trajectory of cells with different deformability in the spiral channel at a flow rate of 1.7 mL / min, with a scale bar of 150 μm; B is a schematic diagram of the deformation-dependent focusing and distribution of cells in the micropillar-assisted spiral channel.

[0022] Figure 3 The following images are used to evaluate the chip sorting performance of different soft and hard cells in this invention: A is a fluorescence image of cancer cells (including GA-fixed cells and CytoD-treated cells) flowing out from different outlets, scale bar 100 μm; B is a statistical analysis of the percentage of GA-fixed cells and CytoD-treated cells at different outlets; C is an image of live / dead cells of MDA-MB-231 cells after passing through the chip, scale bar 100 μm; D is a statistical analysis of cell survival rate after chip sorting of MDA-MB-231 cells; E is the Young's modulus of MDA-MB-231 cells flowing out from different outlets.

[0023] Figure 4To evaluate the sorting of MDA-MB-231 breast cancer cells using the microfluidic chip of this invention; in the figure, A is the fluorescence image of F-actin expression in MDA-MB-231 subpopulations with different stiffness, and the white dashed line represents the cell width and height; B is the aspect ratio of MDA-MB-231 cells under different stiffness; C is the anisotropy of F-actin in BT 549 cells with different stiffness; D is the fluorescence image of F-actin and Vimentin expression in MDA-MB-231 cells with different stiffness; E is the expression of E-cadherin in MDA-MB-231 cells with different stiffness; F is the expression of Vimentin in MDA-MB-231 cells with different stiffness; G is the fluorescence image of MDA-MB-231 cells with different stiffness passing through the Transwell membrane, scale bar 200 μm; H is the quantitative analysis of cancer cells with different deformability passing through the Transwell membrane after 96 h; I is the image of the invasive ability of cancer cells with different deformability after 96 h, scale bar 200 μm.

[0024] Figure 5 To evaluate the sorting of BT-549 breast cancer cells using the microfluidic chip of this invention; in the figure, A is the fluorescence image of F-actin expression in BT 549 subpopulations with different stiffness, and the white dashed line represents the cell width and height; B is the aspect ratio of BT 549 cells with different stiffness; C is the statistical data showing the anisotropy of F-actin in BT 549 cells with different stiffness; D is the fluorescence image of F-actin and Vimentin expression in BT-549 cells with different stiffness; E is the statistical data of E-cadherin expression in BT-549 cells with different stiffness; F is the expression of Vimentin in BT-549 cells with different stiffness; G is the fluorescence image of BT-549 cells with different stiffness passing through the Transwell membrane, scale bar 200 μm; H is the quantitative analysis of cancer cells with different deformability passing through the Transwell membrane after 96 h; I is the image of the invasive ability of cancer cells with different deformability after 96 h, scale bar 200 μm.

[0025] Figure 6 This invention provides real-time cell morphology parameter analysis for the microfluidic chip. A is a schematic diagram of extracting cell features from an image; B is a physical phenotypic method for cell deformation analysis, with a scale bar of 150 μm; C is the quantification of physical parameters of MDA-MB-231 cells, including cell area and cell parameters; D is a scatter plot of MDA-MB-231 cell deformation versus cell area.

[0026] Explanation of the attached diagram labels: 1 is the spiral flow channel, 2 is the micro-column, 3 is the inlet, 4 is the first outlet, and 5 is the second outlet. Detailed Implementation

[0027] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific 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. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0028] Example 1: A microstructured helical inertial microfluidic chip for sorting cell deformability.

[0029] 1. Chip structure design This embodiment is an inertial microfluidic chip with a single helical structure. The main body is a helical channel layer made of PDMS material, fabricated by soft photolithography. The structure is shown in [see attached image]. Figure 1 The spiral channel 1 has a single spiral configuration with 5 circumferences. The cross-section is 50 μm high and 900 μm wide, with an initial radius of curvature of 3 mm and a wall spacing of 300 μm between adjacent circumferences. Micropillars 2 are spaced apart along the inner wall of the spiral channel 1 in the direction of its extension. The central angle between adjacent micropillars 2 and the center point of the spiral channel is π / 6. A total of 12 micropillars 2 are arranged in each circumference. Each micropillar 2 is composed of a cuboid and a hemisphere, where the cuboid is 150 μm long and 200 μm wide, and the hemisphere is 200 μm in diameter. An inlet 3 is provided at the upstream end of the spiral channel 1, and a first outlet 4 and a second outlet 5 are provided at the downstream end. The first outlet 4 and the second outlet 5 are arranged in parallel. The width of the first outlet 4 is 150 μm, and the width of the second outlet 5 is 300 μm.

[0030] 2. Chip fabrication process 2.1 Mask and Master Template Preparation The two-dimensional structure of the chip was drawn using AutoCAD software. After the drawing was completed, the photomask was prepared by Shenzhen MicroChem Optoelectronics Co., Ltd. SU-8 2050 photoresist (MicroChem Corp., USA) was used to prepare a photoresist film on a 4-inch silicon wafer via spin coating at a speed of 2000 rpm, resulting in a film thickness of approximately 80 μm. Pre-baking was performed at 65℃ / 5 min and 95℃ / 15 min. Exposure was performed using a UV lithography machine (365 nm, approximately 180 mJ / cm²), followed by post-exposure baking (PEB) at 65℃ / 2 min and 95℃ / 7 min. Development involved immersion in SU-8 dedicated developer for 8 min, rinsing with isopropanol, and drying with nitrogen. After hardening treatment (150℃, 10 min), the SU-8 positive mold with spiral channel 1 and micropillar 2 protrusions was obtained.

[0031] 2.2 Preparation of the spiral pipe layer Wrap the container holding the master template with aluminum foil to ensure a smooth and flush fit between the template and the bottom of the container. Mix the PDMS prepolymer (Sylgard 184A, Dow Corning) and its matching curing agent (Sylgard 184B, Dow Corning) at a mass ratio of 10:1, stir thoroughly (mixing in a mixer for 2 minutes), then pour the mixture into the container holding the master template. Place the container in a vacuum chamber and evacuate for 45 minutes to completely remove air bubbles from the adhesive. Remove the mixture and place it in an 80°C drying oven for curing for 30 minutes. After curing, peel the PDMS sheet off the master template, cut it along the flow channel contour, and use a punch to machine through holes for inlet 3, first outlet 4, and second outlet 5. Remove dust and impurities from the PDMS surface with tape and set aside.

[0032] 2.3 Preparation of glass slide substrate Prepare PDMS prepolymer and curing agent at a mass ratio of 15:1 and mix thoroughly (mix in a spin coater for 2 min). Fix a clean glass slide onto the spin coater suction cup and add 1 to 2 drops of the mixed adhesive to the center of the slide; set the spin coater program: low speed 500 rpm for 30 s, high speed 1500 rpm for 40 s. After spin coater operation, transfer the glass slide to an 80℃ drying oven for curing for 30 min.

[0033] 2.4 Bonding and Packaging The PDMS channel layer and a clean glass slide were placed together in a plasma cleaner (VP-RS8, Shanzhun) and surface activated at maximum power (13.56 MHz) for 90 s. After the activation, the channel surface of the PDMS channel layer was immediately aligned and pressed with the glass slide to complete the irreversible bonding. The slide was then placed in an 80°C oven for 3 days for heat treatment.

[0034] Example 2: Chip-based sorting and functional verification of the deformability of MDA-MB-231 breast cancer cells.

[0035] 1. Experimental Materials and Cell Culture Cell line: Human triple-negative breast cancer cell line MDA-MB-231, purchased from the American Type Culture Collection (ATCC).

[0036] Culture conditions: DMEM medium supplemented with 10% fetal bovine serum (FBS) + 1% penicillin-streptomycin was used and cultured in a saturated humidity incubator at 37°C and 5% CO2. When the cell confluence reached 90%, cells were digested and passaged using TrypLE. Cells were passaged every 2 to 3 days, and cells in the logarithmic growth phase were used for experiments.

[0037] Main reagents and equipment: PBS buffer, bovine serum albumin (BSA), microinjection pump, atomic force microscope (NanoWizard 4), inverted fluorescence microscope, etc.

[0038] 2. Cell sorting operation Digested MDA-MB-231 cells were resuspended in PBS buffer containing 0.1% BSA to adjust the cell concentration to 1.2 × 10⁻⁶ cells / mL. 6 Cells / mL; the cell suspension was injected uniformly from the chip inlet at a flow rate of 1.7 mL / min using a syringe pump.

[0039] Within the spiral flow channel, cells migrate laterally via Dean's secondary flow regulated by microcolumns: hard cells (low deformability) focus on the flow channel region near the inner wall and are collected from the first outlet 4; soft cells (high deformability) focus on the flow channel region away from the inner wall and are collected from the second outlet 5. Figure 2 (A and B). To verify the sorting purity of this invention for cells with different deformities, GA-fixed cells (simulating hard cells) and CytoD-treated cells (simulating soft cells) were used as models. The two types of cells were mixed and then sorted. The results showed that most of the cells collected at the first outlet 4 were GA-fixed hard cells, while most of the cells collected at the second outlet 5 were CytoD-treated soft cells. Figure 3 (A) Quantitatively counted the number of cells at the first outlet 4 and the second outlet 5, and the results are as follows: Figure 3 As shown in B; and the cell viability remained above 90% after sorting. Figure 3 The cells collected (C and D) were used for subsequent mechanical characterization and biological function experiments, respectively.

[0040] 3. Verification of the mechanical properties of sorted cells (atomic force microscopy indentation test) To accommodate single-cell atomic force microscopy (AFM) indentation testing, a microporous array with a diameter of 20 μm and a depth of 10 μm was designed. This ensures that each well can precisely accommodate one suspended breast cancer cell, allowing the AFM probe to contact the top of the cell from above. Fabrication employed a soft lithography and NOA (Noise- and Acid-curing Adhesive) molding process: First, a positive mold of micropillars 2 was fabricated using SU-8 lithography. Then, PDMS was poured in and cured to obtain a stamp with the raised micropillars 2. This PDMS stamp was then inverted onto a glass slide, and NOA adhesive was introduced via capillary action to fill the gap between the stamp and the glass. The adhesive layer was then cured by UV irradiation (50 mW / cm², 1 min). Finally, the PDMS stamp was peeled off, leaving indentations on the glass slide corresponding to the micropillars 2, forming the microporous array. To prevent non-specific cell adhesion to the well walls, the microporous chip was treated with 0.2% Pluronic solution for 20 min to form an anti-adsorption layer on the well surface, ensuring the cells remain "suspended" for interference-free AFM measurements. Before testing, cells were suspended in serum-free culture medium, seeded into a microarray, and left to stand for 15 minutes to allow single cells to settle naturally into the wells by gravity, thus completing single-cell capture.

[0041] Indentation tests were performed using an atomic force microscope equipped with a 4.5 μm polystyrene microsphere probe. The probe spring constant was 0.03 N / m, the maximum applied force was 3 nN, and the loading rate was 5 μm / s. Ten cells were tested in each group, and 25 force-distance curves were collected for each cell. The Young's modulus of the cell was calculated using a Hertzian contact model.

[0042] The results showed that the average Young's modulus of cells collected at the first outlet 4 was approximately 600 Pa (hard cell subset), while the average Young's modulus of cells collected at the second outlet 5 was approximately 300 Pa (soft cell subset). Figure 3 (E), proving that this chip can achieve effective sorting based on cell deformability.

[0043] 4. Verification of biological functions of cells after sorting The expression of cytoskeleton and metastasis-related proteins and their migration and invasion capabilities were detected in two cell subpopulations: 4.1 Immunofluorescence After removing the culture medium, wash 3 times with PBS; fix with 4% paraformaldehyde for 15 min, wash 3 times with PBS; permeabilize with 0.3% Triton X-100 for 10 min; block with 5% BSA for 1 h. Cytoskeleton staining: add Phalloidin-iFluor 647, incubate at 4℃ for 2 h, wash 3 times with PBS (5 min each time). EMT protein staining: add rabbit anti-E-cadherin (Bioss, bs-10009R) and mouse anti-Vimentin (Bioss, bsm-33170M) primary antibodies, incubate overnight at 4℃; after washing 3 times with PBS, add Alexa Fluor 488-labeled anti-rabbit secondary antibody (Bioss, bs-0295G-BF488) and Alexa Fluor 647-labeled anti-mouse secondary antibody (Bioss, bs-0296G-BF647), incubate at 37℃ in the dark for 2 h; wash 3 times with PBS. Imaging using a confocal microscope (Nikon A1R HD25).

[0044] The results showed that the soft cell subset had a larger cell length-to-width ratio ( ). Figure 4 A, B), F-actin anisotropy decreased ( Figure 4 The C-type cells exhibit mesenchymal-like morphology and cytoskeleton remodeling characteristics. E-cadherin expression in soft cell subsets is significantly downregulated. Figure 4 D and E), and the expression of vimentin was significantly upregulated ( Figure 4 The D and F values ​​conform to the molecular phenotype of cells with high metastatic potential.

[0045] 4.2 Detection of cell migration and invasion capabilities Migration ability assay: More deformable and less deformable cancer cell subpopulations obtained by microfluidic sorting were resuspended in serum-free medium and seeded into the upper chamber of a Transwell chamber. The lower chamber was incubated with complete medium containing 10% fetal bovine serum as a chemical inducer. The chambers were incubated at 37°C and 5% CO2 for 48 h. After incubation, any unmigrated cells remaining on the upper chamber membrane were gently wiped away with a cotton swab. The chambers were then fixed in 4% paraformaldehyde at room temperature for 15 min. After washing with PBS, the cells were stained with 0.1% crystal violet solution at 25°C for 20 min. Excess dye was washed away with PBS, and the cells were air-dried. The number of cells that migrated to the lower membrane was counted in multiple randomly selected fields of view under a microscope to reflect the in vitro migration ability of different deformable subpopulations.

[0046] Invasion assay: A layer of Matrigel substrate was pre-coated onto the membrane surface of the upper chamber of the Transwell chamber to simulate the extracellular matrix barrier. Different deformed cell subpopulations were seeded into the upper chamber, while the lower chamber was placed in complete culture medium containing 10% fetal bovine serum as a chemical inducer. The cells were incubated at 37°C with 5% CO2 for 96 h to allow time for Matrigel degradation and cell penetration. After incubation, residual cells and Matrigel layer in the upper chamber were carefully wiped away. The chamber was then fixed in 4% paraformaldehyde at room temperature for 15 min, washed with PBS, and stained with 0.1% crystal violet solution at room temperature for 20 min. Excess dye was washed away with PBS, and the cells were air-dried. Cells were counted under a microscope to evaluate the invasive ability of each subpopulation after overcoming physical and enzymatic barriers.

[0047] The results showed that the in vitro migration and invasion capabilities of the soft cell subset were 2 to 3 times that of the hard cell subset, verifying the negative correlation between cell deformability and metastasis potential. Figure 4 (G, H and I).

[0048] Example 3: Verification of the universality of sorting the BT-549 breast cancer cell line.

[0049] Following the experimental procedure of Example 2, the human triple-negative breast cancer cell line BT-549 was sorted and its function was verified.

[0050] The results showed that the sorting pattern of BT-549 cells was highly consistent with that of MDA-MB-231 cells—the cell aspect ratio was increased ( Figure 5 The anisotropy of A, B) and F-actin is reduced ( Figure 5 (C), while also showing upregulation of vimentin expression ( Figure 5 Downregulation of D, F) and E-cadherin expression ( Figure 5 The migration ability of the soft cell subset (second exit 5) was 2.5 times that of the hard cell subset (D, E). Figure 5 (G, H, and I). This result demonstrates that the deformability sorting strategy of this chip is universal in different breast cancer cell lines.

[0051] Example 4: Image-based real-time cell deformation phenotypic analysis.

[0052] A high-speed imaging system was built in the chip exit area. A Fastcam SA3 camera (Photron, Japan) mounted on an inverted microscope was used to acquire real-time bright-field images of the flowing cells, with an imaging frame rate of 100 fps to 7000 fps. Image-Pro Plus software was used to automatically identify individual cells and extract morphological parameters such as area, perimeter, and roundness. The deformability index was defined as 1 - cell roundness, where cell roundness = It is used to quantitatively assess the deformability of cells.

[0053] The results showed that the average cell area and perimeter of the hard cell subpopulation at the first outlet 4 were both greater than those of the soft cell subpopulation at the second outlet 5. Figure 6 (A, B); the proportion of cells with high deformability index in the soft cell subset was significantly higher than that in the hard cell subset ( Figure 6 (C, D). This method can obtain cell mechanics-related morphological phenotypes without fluorescent labeling and can be linked with the sorting process to achieve online quality control.

[0054] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments.

[0055] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A microstructured helical inertial microfluidic chip for sorting cell deformability, characterized in that, It includes a spiral flow channel (1), on which a number of micro-pillars (2) are arranged at equal intervals along the length of the spiral flow channel (1) on the inner sidewall facing the center of curvature; the upstream end of the spiral flow channel (1) is provided with an inlet (3), and the downstream end is provided with a first outlet (4) and a second outlet (5). The micro-column (2) is integrally formed by connecting the lower cuboid section and the upper semi-circular arch section. The straight bottom edge of the semi-circular arch section is completely attached to the upper surface of the cuboid section, and the overall top surface is raised in a smooth arc within the spiral flow channel (1).

2. The microstructured helical inertial microfluidic chip according to claim 1, characterized in that, The spiral channel (1) is a planar single spiral configuration with 4 to 6 spiral turns and a spiral spacing of 200 μm to 400 μm; the cross-sectional height is 40 μm to 60 μm, the width is 800 μm to 1000 μm, and the initial radius of curvature is 2 mm to 4 mm.

3. The microstructured helical inertial microfluidic chip according to claim 1, characterized in that, The central angle between adjacent microcolumns (2) and the center point of the spiral channel (1) is π / 8 to π / 4, and a total of 10 to 15 microcolumns (2) are set every week.

4. The microstructured helical inertial microfluidic chip according to claim 3, characterized in that, The cuboid segment has a length of 300 μm to 400 μm, a width of 150 μm to 250 μm, and a height of 40 μm to 60 μm; the semi-circular arch segment has a diameter of 150 μm to 250 μm and a height of 40 μm to 60 μm.

5. The microstructured helical inertial microfluidic chip according to claim 1, characterized in that, The first outlet (4) has a cross-sectional width of 100 μm to 200 μm and a cross-sectional height of 40 μm to 60 μm; the second outlet (5) has a cross-sectional width of 200 μm to 400 μm and a cross-sectional height of 40 μm to 60 μm.

6. The application of the microstructured helical inertial microfluidic chip according to any one of claims 1 to 5 in sorting cells of different hardness.

7. The application according to claim 6, characterized in that, Specifically, the steps include: The cells to be sorted were resuspended in PBS buffer containing bovine serum albumin and the cell concentration was adjusted to 1.0 × 10⁶ cells / day. 6 cells / mL ~1.5×10 6 cells / mL; The cell suspension was injected at a constant rate of 1.2 mL / min to 2.0 mL / min from the inlet (3) of the spiral channel (1); Cells migrate laterally within the spiral channel (1) under the Dean secondary flow enhanced by micropillars (2). Hard cells with weak deformability focus on the channel area near the inner wall and are collected from the first outlet (4); soft cells with strong deformability focus on the channel area away from the inner wall and are collected from the second outlet (5), thus completing the sorting of cells with different hardness.

8. The application according to claim 7, characterized in that, It also includes a real-time cell morphology phenotypic analysis step: a high-speed imaging system is built in the outlet area of ​​the spiral channel (1) to collect real-time bright field images of the flowing cells, automatically identify individual cells and extract cell area, perimeter and roundness morphological parameters, and quantify cell deformation ability through the deformation ability index to achieve online quality control of the sorting process.

Images