Microfluidic chip, system and application for cell separation

Abstract

The application provides a microfluidic chip, system and application for cell separation, and belongs to the technical field of microfluidic chips. The microfluidic chip comprises a chip body; a liquid inlet channel, a non-blocking microfluidic channel and a liquid storage pool in communication with an outlet end of the microfluidic channel are arranged in the chip body, and the inner wall of the microfluidic channel is modified with P-selectin for capturing target cells; the microfluidic channel is an integrated anti-bubble structure, the anti-bubble structure comprises a neck portion, an expansion section and an extension section in sequence along the fluid flow direction, and the extension section is connected to the liquid storage pool. The application can effectively avoid bubble formation and improve the recovery efficiency of target cells.

Description

Technical Field

[0001] This application relates to the field of microfluidic chip technology, and in particular to a microfluidic chip, system and application for cell separation. Background Technology

[0002] Tumor metastasis is a critical stage in the development of cancer. Non-metastatic and highly metastatic tumor cells / spheroids differ significantly in their biological behavior, invasiveness, and drug sensitivity. Achieving efficient and precise separation of these two types of cells is crucial for research into tumor metastasis mechanisms, screening of anti-tumor drugs, and personalized cancer treatment. Microfluidic technology, with its advantages of miniaturization, high throughput, low sample consumption, and the ability to simulate the in vivo physiological microenvironment, has become an important technique in tumor cell separation. Various microfluidic platforms have been developed for the separation of both non-metastatic and highly metastatic cells / spheroids.

[0003] Existing microfluidic platforms primarily rely on biomechanical capture mechanisms to separate metastatic cells. For example, patents CN109852544A and CN107402295A disclose methods that employ physical structures such as asymmetric mechanical ratchet arrays and confined hydraulic channels for cell capture, or quantify cell transport capacity through contractile deformability measurements to achieve separation and screening. While these methods have shown some effectiveness in selectively capturing invasive cells, they suffer from unavoidable technical drawbacks: their separation principle depends on complex channel geometry designs. Such structures easily disrupt the laminar flow state in the microfluidic system, significantly increasing the difficulty of chip manufacturing and integration, and creating conditions for bubble formation, severely affecting the stability and reproducibility of the separation process.

[0004] Bubble formation is a common and critical challenge in microfluidic technology applications, particularly prominent in microfluidic devices for the separation of metastatic cells / spheres. Existing microfluidic chips possess geometrically confined structures, such as microwells, acute channel angles, contact areas of assembled components, and rough geometries like micropillars, which serve as primary sites for bubble nucleation. Bubbles readily form during chip surface coating and experimental initiation. Simultaneously, structures like micropillars create minimum pressure zones within the flow channels, further promoting bubble formation and aggregation. These formed bubbles severely distort laminar shear stress, causing irregular fluctuations in shear stress and pressure. This not only leads to the detachment of adhered tumor cells from the chip channel surface but also disrupts the normal fluidic trajectory of cells / spheres, significantly reducing the reproducibility of adhesion assays and cell separation, and impacting the accuracy of separation results.

[0005] In summary, existing microfluidic technologies for separating non-metastatic and highly metastatic cells / spheres suffer from low separation efficiency, poor reproducibility, and insufficient cell viability due to complex channel design and difficulty in suppressing bubble formation, making them unsuitable for practical applications in tumor metastasis research. Developing a microfluidic chip with a simple channel design that effectively suppresses bubble formation has become a pressing technical problem in this field. Summary of the Invention

[0006] The purpose of this application is to provide a microfluidic chip, system, and application for cell separation to solve at least one of the above-mentioned technical problems.

[0007] In a first aspect, this application provides a microfluidic chip for cell separation, comprising: a chip body; The chip body has a liquid inlet channel, a non-blocking microfluidic channel, and a liquid reservoir connected to the outlet end of the microfluidic channel. The inner wall of the microfluidic channel is modified with P-selectin for capturing target cells. The microfluidic channel is an integrated anti-bubble structure, which includes a neck, an extension section, and an extension section in sequence along the fluid flow direction. The extension section is connected to the liquid storage tank.

[0008] Optionally, the liquid inlet channel extends from the upper surface of the chip body into the interior of the chip body and is smoothly connected to the inlet end of the microfluidic channel; The microfluidic channel extends inside the chip body and is smoothly connected to the liquid reservoir at the outlet end. The liquid reservoir extends from inside the chip body out of the upper surface of the chip body.

[0009] Optionally, the upper end face of the liquid storage tank is a circular planar structure, used to connect the upwardly extending Luer joint to form a liquid storage section.

[0010] Optionally, the liquid storage tank and the outlet end of the microfluidic channel are connected by an arc transition, and the radius of curvature of the transition arc is adapted to the requirement of non-turbulent fluid flow.

[0011] Optionally, the cross-section of the extension is rectangular, and the width of the extension is 3~5mm, the height is 200~300μm, and the length is 30~80mm.

[0012] Optionally, the ratio of the length:width:height of the extension segment is in the range of (120~300):(12~30):1.

[0013] Optionally, the extension section is a tapered extension section, and the angle between the side of the tapered extension section and the fluid flow direction of the extension section is 30° to 60°.

[0014] Optionally, the width of the neck is 0.8 to 1.2 mm, and the width of the extension is 3 to 5 mm.

[0015] In a second aspect, this application provides a cell separation system, the system comprising at least one microfluidic chip as described in any embodiment of this application, and a pressure control component sealed and connected to the microfluidic chip; The pneumatic control component includes an injection pump and a pneumatic transmission pipeline. The first end of the pneumatic transmission pipeline is sealed to the upper end face of the liquid storage tank, and the second end opposite to the first end is connected to the injection pump. A pneumatic controller is configured inside the pneumatic transmission pipeline.

[0016] Optionally, the microfluidic chip comprises N chips, where N is a positive integer greater than or equal to 2; The number of first ends of the air pressure transmission pipeline is the same as the number of microfluidic chips, and each first end is sealed and connected to each microfluidic chip in a one-to-one correspondence. The pneumatic transmission pipeline has a step-by-step converging structure, including sub-segments connected to each first end. Multiple sub-segments merge to form a first-level converging segment, and each level of converging segment merges upwards in sequence, finally converging to the second end. Each sub-segment and merging segment is equipped with a pressure controller.

[0017] In a third aspect, this application provides the application of a microfluidic chip or cell separation system as described in any embodiment of this application in cell separation, for separating target cells and non-target cells.

[0018] The microfluidic chip, system, and application for cell separation described in this application feature an integrated anti-bubble structure in the microfluidic channel with virtually no bubble nucleation sites during the separation of target cells. This effectively prevents bubble formation, ensures the stability of laminar shear stress, and prevents adhered target cells from detaching due to shear stress fluctuations caused by bubbles. The non-blocking channel structure avoids cell / sphere retention and blockage, significantly improving the efficiency, repeatability, and biological fidelity of cell separation, and enhancing the recovery efficiency of target cells. Attached Figure Description

[0019] To more clearly illustrate the technical solutions of the embodiments of this application, the accompanying drawings used in the embodiments will be briefly described below. It should be understood that the following drawings only show some embodiments of this application and should not be regarded as a limitation on the scope of this application.

[0020] Figure 1 This is a top view of the fluid channels of a microfluidic chip in one embodiment; Figure 2 This is a side view of some components of a microfluidic chip in one embodiment; Figure 3 This is a schematic diagram of the cell separation system in one embodiment; Figure 4 This is a schematic diagram of the dimensions of the fluid channels of a microfluidic chip in one embodiment.

[0021] Explanation of reference numerals: 110, Chip body; 120, Liquid inlet channel; 130, Microfluidic channel; 140, Liquid reservoir; 131, Neck; 132, Extension section; 133, Extension section; 210, Luer connector; 220, Sample injection assembly; 230, Pressure controller; 240, Sub-segment; 250, Merging section. Detailed Implementation

[0022] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application.

[0023] All terms used in this application (including technical and scientific terms) have the meanings commonly understood by those skilled in the art, unless otherwise defined. It should be noted that the terms used herein should be interpreted in a manner consistent with the context of this specification, and not in an idealized or overly rigid way.

[0024] For example, the terms "first," "second," etc., used in this application may be used herein to describe various elements, but these elements are not limited by these terms. These terms are only used to distinguish the first element from another element.

[0025] For example, the terms "comprising" or "including" used in this application indicate the presence of features, steps, operations and / or components, but do not exclude the presence or addition of one or more other features, steps, operations or components.

[0026] Combination Figure 1 and Figure 2 As shown, this application provides a microfluidic chip for cell separation, comprising: a chip body 110; the chip body 110 having an inlet channel 120, a non-blocking microfluidic channel 130, and a reservoir 140 communicating with the outlet end of the microfluidic channel 130; the inner wall of the microfluidic channel 130 being modified with P-selectin for capturing target cells; the microfluidic channel 130 having an integrated anti-bubble structure, the anti-bubble structure including, along the fluid flow direction, a neck 131, an extension section 132, and an extension section 133, the extension section 133 being connected to the reservoir 140.

[0027] In this embodiment, target cells refer to cells and / or cell aggregates that need to be specifically captured by a microfluidic chip. For example, the target cells may be highly metastatic tumor cells and / or spheroids, which differ significantly from non-target cells (non-metastatic tumor cells and / or spheroids) in their bioadhesion characteristics, making them the capture targets of the microfluidic chip. Specifically, target cells may include highly metastatic cells such as HEYA8 HM, Kuramochi, and SKOV3 M-CSC.

[0028] P-selectin refers to a membrane glycoprotein cell adhesion molecule (also known as CD62P or GMP140) belonging to the selectin family. Its molecular structure includes core ligand-binding sites such as a lectin-like region and an epidermal growth factor-like region. It can specifically bind to ligands such as S-Lewis X antigen and PSGL-1, which are highly expressed on the surface of target cells such as highly metastatic tumor cells / spheroids, making it a core biomolecule for achieving specific capture of target cells. P-selectin exhibits excellent biological specificity; non-target cells that do not specifically bind to it cannot form stable adhesion, ensuring precise cell separation. Specifically, a P-selectin solution at a concentration of 20–30 μg / mL can be used to coat the inner wall of microfluidic channel 130, allowing it to specifically bind to target cells and achieve their capture.

[0029] The chip body 110 refers to the basic substrate structure of the microfluidic chip. It is the carrier of microstructures such as the liquid inlet channel 120, microfluidic channel 130, and liquid reservoir 140. Its material must meet the requirements of biocompatibility, microfabrication and hydrophilicity (or modifiable hydrophilicity) to provide a stable physical substrate for cell separation.

[0030] The inlet channel 120 refers to a microchannel structure formed inside the chip body 110 for delivering a suspension containing target cells to the microfluidic channel 130. It is a sample introduction pathway for cell suspension. One end forms an inlet adapted to the conventional sample introduction component 220, and the other end is smoothly connected to the microfluidic channel 130 to ensure that the fluid enters the microfluidic channel 130 at a uniform speed without stagnation.

[0031] The non-blocking microfluidic channel 130 refers to a micron-sized channel formed within the chip body 110, without any protrusions, depressions, corners, or geometric traps. It is the core functional area for cell separation. Its inner wall is modified with P-selectin to capture target cells. Simultaneously, the non-blocking structure avoids fluid turbulence, bubble formation, and cell / sphere retention, ensuring separation efficiency. The reservoir 140 is a cavity structure formed within the chip body 110 and connected to the outlet of the microfluidic channel 130. It is the cell collection area of ​​the microfluidic chip, primarily used to collect non-target cells that have not adhered to P-selectin. Its smooth connection to the microfluidic channel 130 enables direct, lossless cell collection, avoiding secondary transport losses in downstream pipes / components.

[0032] The integrated anti-bubble structure refers to the microfluidic channel 130 being a single, integrally molded structure with smooth transitions between segments, free of sharp angles and / or dead angles. Along the fluid flow direction, it sequentially includes a neck 131, an extension segment 132, and an extension segment 133. This structure eliminates bubble nucleation sites at the source, preventing bubble formation and aggregation during microfluidic processes and ensuring the stability of laminar shear stress. For example, the neck 131, extension segment 132, and extension segment 133 of the microfluidic channel 130 are integrally formed using soft photolithography, with rounded transitions between segments, eliminating any sharp angles, dead angles, or rough geometric structures, effectively preventing bubble nucleation and aggregation within the channel.

[0033] The neck 131 refers to the initial section near the inlet channel 120 in the integrated anti-bubble structure. It serves as the guide section for the microfluidic channel 130, ensuring a stable fluid flow transition from the inlet channel 120 to the extension section 132. Its dimensions are adapted to both the inlet channel 120 and the extension section 132, guaranteeing that the fluid enters the subsequent channel structure without turbulence. The extension section 132 is the transition section connecting the neck 131 and the extension section 133 in the integrated anti-bubble structure. It is a diameter-expanding structure that allows for a smooth expansion of the fluid from a narrow to a wide channel, avoiding turbulence, bubbles, and cell / sphere impact and retention caused by sudden channel expansion. It is a key structure for anti-bubble and stable flow. The extension section 133 is the main section connecting the extension section 132 and the reservoir 140 in the integrated anti-bubble structure. It is a non-blocking straight channel and the core region for P-selectin-mediated target cell capture. It has sufficient length and cross-sectional area to provide ample effective contact area between the target cells and the P-selectin on the channel wall, ensuring the target cell capture efficiency.

[0034] Specifically, the liquid inlet channel 120 is a cylindrical through-hole structure, which is opened on the upper part of one side of the chip body 110, extending vertically from the upper end face of the chip body 110 into the interior of the chip body 110, and the lower end is connected to the neck 131 of the microfluidic channel 130 by a rounded transition; the inner diameter of the liquid inlet channel 120 is 1.5mm±0.5mm, and the total length can be 8mm±2mm. Its upper end forms a funnel-shaped sample inlet to be compatible with conventional microfluidic sample introduction components 220 such as P200 pipette tips and micro-injection needles, so as to ensure that suspensions such as cells / spheres can be injected into the microfluidic channel 130 at a uniform speed and without retention.

[0035] For example, the inlet channel 120 has an inner diameter of 1.5 mm and a total length of 8 mm. Its upper end forms a funnel-shaped inlet, which is compatible with conventional microfluidic injection components 220 such as P200 pipette tips and micro-injection needles, ensuring that cell / sphere suspensions can be injected into the microfluidic channel 130 at a uniform speed without retention.

[0036] The non-blocking microfluidic channel 130 is an integrated anti-bubble structure, horizontally opened inside the chip body 110, and vertically connected to the liquid inlet channel 120. It has no protrusions, depressions, or corners throughout, and each section is connected by a rounded transition (for example, the radius of curvature of the transition arc is 1mm). There are no sharp corners, dead corners, or other bubble nucleation sites. Along the fluid flow direction, there are a neck 131, an extension section 132, and an extension section 133, all of which have rectangular cross-sections.

[0037] The inner wall of microfluidic channel 130 is modified with P-selectin. The modification method is as follows: P-selectin phosphate buffer (PBS, pH=6.5) at a concentration of 20~30 μg / mL is injected into microfluidic channel 130 to fill the entire channel. After sealing the inlet and outlet, it is incubated at a constant temperature of 4℃ for 12h. The coating method can be to first add protein A for physical adsorption coating, and then add recombinant P-selectin-Fc protein for coating. The channel is washed three times with PBS buffer to remove unadsorbed free P-selectin, and finally a P-selectin modified layer is formed to achieve specific adhesion to target cells.

[0038] The reservoir 140 is a cylindrical cavity structure located on the side of the chip body 110 away from the inlet channel 120. It is connected to the outlet end of the extension 133 of the microfluidic channel 130 with a rounded transition (the radius of curvature of the transition arc is 2 mm). It extends from the outlet end of the microfluidic channel 130 towards the upper surface of the chip body 110, and the upper surface of the reservoir 140 protrudes beyond the upper surface of the chip body 110 (e.g., protruding 2 mm), forming an open collection cavity. The reservoir 140 has an inner diameter of 4 ± 1 mm, a depth of 8 ± 2 mm, and an effective volume of 100 ± 20 μL. It can directly collect non-target cells / spheres flowing out of the microfluidic channel 130, avoiding secondary transport losses in downstream pipelines or syringes. The upper surface of the reservoir 140 is used to connect an external pneumatic control component via a Luer connector to achieve pneumatically driven fluid flow. The reservoir 140 allows for liquid extraction while retaining the suspension, avoiding long-term cell storage in the syringe.

[0039] When the microfluidic chip of this application is used for target cell separation, the operation process and core principle are as follows: Sample introduction: The prepared suspension containing target cells is injected into the inlet channel 120 through the P200 pipette tip. Under the drive of gravity and subsequent gas pressure, the suspension flows smoothly into the neck 131 of the microfluidic channel 130 through the inlet channel 120. After being guided by the neck 131, the suspension enters the conical extension section to achieve smooth expansion. The fluid always maintains a laminar flow state, without turbulence or bubble formation. After the laminar flow suspension enters the extension section 133, under the fluid environment of physiological shear stress (0.01~0.1 dyn / cm²), the target cells (such as highly metastatic tumor cells and / or spheroids) are separated. Because of the high expression of P-selectin ligands such as S-Lewis X antigen on its surface, it undergoes specific and stable bioadhesion with the P-selectin on the inner wall of the extension segment 133, achieving precise capture of target cells. Non-target cells, because they hardly express P-selectin ligands on their surface, cannot form effective adhesion with the P-selectin on the inner wall of the channel, and continue to flow along the extension segment 133 with the fluid, smoothly entering the reservoir 140 through the outlet end, completing the direct and lossless collection of non-target cells. After separation, PBS buffer containing 2mM EDTA is injected into the microfluidic channel 130, and after incubation for 5 min, the captured target cells are eluted by gentle air pressure, achieving the recovery of target cells.

[0040] Throughout the separation process, the integrated anti-bubble structure of the microfluidic channel has almost no bubble nucleation sites, effectively preventing bubble formation, ensuring the stability of laminar shear stress, and preventing the shear stress fluctuations caused by bubbles from causing the adhering target cells to fall off. The non-blocking channel structure avoids cell / sphere retention and blockage, greatly improving the efficiency, repeatability and biological fidelity of cell separation.

[0041] In one embodiment, combined Figure 2As shown, the liquid inlet channel 120 extends from the upper end face of the chip body 110 into the interior of the chip body 110 and is smoothly connected to the inlet end of the microfluidic channel 130; the microfluidic channel 130 extends inside the chip body 110 and is smoothly connected to the liquid reservoir 140 at the outlet end; the liquid reservoir 140 extends from the interior of the chip body 110 out of the upper end face of the chip body 110.

[0042] In one embodiment, combined Figure 2 As shown, the upper surface of the liquid storage tank 140 is a circular planar structure, which is used to connect the upwardly extending Luer connector to form a liquid storage section.

[0043] In one embodiment, combined Figure 1 As shown, the outlet end of the liquid storage tank 140 and the microfluidic channel 130 are connected by an arc transition, and the radius of curvature of the transition arc is adapted to the requirement of non-turbulent fluid flow.

[0044] In this embodiment, smooth connection refers to the connection method between the channels / cavities of the microfluidic chip, which adopts a rounded transition, oblique angle transition, or other connection method without sharp angles or dead angles. There are no sudden changes in cross-section or physical protrusions, which ensures that the fluid flows in a laminar state at the structural connection, avoiding turbulence, bubble formation, and retention and blockage of cells / spheres. It is a key structural feature for achieving anti-bubble and low-damage cell separation.

[0045] The inlet channel 120 is a vertically extending cylindrical through-hole structure, extending vertically downwards from the upper surface of the chip body 110 into its interior. For example, its extension depth is 8 mm and its inner diameter is 1.5 mm. The lower end of the inlet channel 120 is smoothly connected to the neck 131 of the microfluidic channel 130 via a quarter-circle arc with a curvature radius of 1 mm. The connecting section is the same width and height as the neck 131, with no abrupt change in cross-section. The upper end of the inlet channel 120 forms a funnel-shaped injection port, compatible with conventional microfluidic injection components 220 such as P200 pipette tips and micro-injection needles, ensuring uniform and non-residual injection of cell suspension. The outlet end of the microfluidic channel 130 is smoothly connected to the reservoir 140 via an arc with a curvature radius of 2 mm, ensuring no sudden changes in flow velocity or eddy currents at the connection point.

[0046] The liquid inlet channel 120 is a vertical straight-through structure, extending vertically downward from the upper end face of the chip body 110 into the interior of the chip body 110, with an extension depth of 8mm (the total height of the chip body 110 is 5mm, and the lower end of the liquid inlet channel 120 extends to the inner side of the bottom of the chip body 110 by 3mm). The lower end of the liquid inlet channel 120 bends horizontally towards the interior of the chip body 110 and is smoothly connected to the inlet end (the starting end of the neck 131) of the microfluidic channel 130 through a quarter-circle arc structure with a curvature radius of 1mm. The arc connection section has the same width and height (1mm width, 250μm height) as the neck 131 of the microfluidic channel 130, with no sudden change in cross-section, ensuring that the vertically flowing cell suspension smoothly transitions to horizontal flow into the microfluidic channel 130 after the arc transition, avoiding fluid impact that generates turbulence and bubbles.

[0047] The microfluidic channel 130 is a horizontal, linear, non-blocking structure that extends linearly in the horizontal direction within the chip body 110, such as... Figure 4 As shown, its total extension length is approximately 46.66 mm (neck length approximately 2.97 mm, inner diameter approximately 0.01 mm, extension section length approximately 2.065 mm, and extension section length approximately 41.63 mm). It remains parallel to the bottom of the chip body 110 throughout its length. The centerline of the channel is approximately 1.5 mm from the bottom of the chip body 110. The angle between the edge of the tapered extension section and the fluid flow direction of the extension section is approximately 37°, ensuring the stability of the channel structure. Combined with the 4 mm width of the extension section and the 2... With a height of 50μm, the flow rate of liquid can be controlled within the range of 8.3~50μL / s. The outlet end of the microfluidic channel 130 (the end of the extension section 133 away from the extension section 132) smoothly transitions to the reservoir 140 with a radius of curvature of 2mm. There is no cross-sectional change at the connection between the transition section and the extension section 133 and the reservoir 140. After the fluid flows out horizontally from the microfluidic channel 130, it smoothly enters the reservoir 140 through the arc transition, avoiding the retention of cells / spheres at the outlet end.

[0048] Specifically, the upper end of the liquid storage tank 140 is equipped with a standard Luer female connector, which can be quickly snapped into the Luer male connector of the external pneumatic transmission pipeline. Pneumatic sealed transmission can be achieved without additional sealing parts, and it is compatible with the integration of multi-channel and multi-path multiplexing pipelines.

[0049] The upper surface of the liquid storage tank 140 is designed as a circular planar structure, which can be connected to an upwardly extending Luer connector as a larger capacity liquid storage part. Specifically, the Luer connector can be a male Luer connector, which is then press-fitted with the male Luer connector to connect to the pneumatic control component. It extends vertically upward from the arc transition section at the outlet end of the microfluidic channel 130 to the upper surface of the chip body 110, and the upper part of the liquid storage tank 140 extends through the upper surface of the chip body 110 to form an independent collection cavity protruding from the chip body 110.

[0050] In this application, the liquid inlet channel 120, microfluidic channel 130, and liquid reservoir 140 are smoothly connected within the chip body 110 without intersection, interference, or sharp angles. The liquid inlet channel 120 is located on the upper left side of the chip body 110, the microfluidic channel 130 extends along the horizontal centerline of the chip body 110, and the liquid reservoir 140 is located on the upper right side of the chip body 110. The three form an integrated spatial layout of left-inlet-middle-separation-right-collection. The fluid flow path is short and without backflow, which greatly reduces the fluid flow resistance, ensures the stability of the laminar flow state, avoids turbulence, bubble formation, and cell / sphere retention, improves separation efficiency and repeatability, and facilitates chip operation, integration, and multiplexing.

[0051] The connection between the reservoir and the Luer connector forms a reservoir section with a larger volume than the reservoir itself. This increases the effective storage volume and provides an independent operating space for external pressure control components and pipelines, avoiding interference between pipeline connections and the chip body 110 and improving the chip's ease of operation. The standardized functional port configuration of the reservoir and Luer connector enables precise and sealed transmission of air pressure, replacing traditional direct fluid drive and reducing cell shear stress damage. At the same time, the universal compatibility of the Luer connector 210 ensures the chip's integration and multiplexing capabilities, bridging the technological gap between microfluidic adhesion detection and large-scale functional analysis.

[0052] In one embodiment, the cross-section of the extension segment 133 is rectangular, and the width of the extension segment 133 is 3~5mm, the height is 200~300μm, and the length is 30~80mm. For example, the width can be 4±0.5mm (e.g., 3.5mm, 3.7mm, 3.8mm, 3.9mm, 4mm, 4.1mm, 4.2mm, 4.3mm, 4.4mm, or any suitable width within the width range formed by these widths), the height can be 250±20μm (e.g., 230μm, 235μm, 240μm, 245μm, 248μm, 250μm, 252μm, 255μm, 260μm, 265μm, 270μm, or any suitable height within the height range formed by these heights), and the length can be 45±5mm (e.g., 40mm, 43mm, 44mm, 45mm, 46mm, 46.5mm, 47mm, 48mm, 50mm, or any suitable length within the length range formed by these lengths).

[0053] Optionally, the length:width:height ratio of the extension segment 133 can be in the range of (120~300):(12~30):1. For example, its length:width:height ratio is (180±20):(15±2):1.

[0054] In one embodiment, the extension segment 132 is a tapered extension segment, and the angle formed between the side of the tapered extension segment and the fluid flow direction of the extension segment 133 is 30° to 60°. Specifically, this angle can be any suitable degree such as 30°, 33°, 35°, 37°, 40°, 45°, 48°, 50°, 52°, 53°, 54°, 55°, 57°, 58°, 60°, or any suitable degree range formed by these degrees.

[0055] In one embodiment, the width of the neck 131 is 0.8~1.2mm, and the width of the extension 133 is 3~5mm. For example, the width of the neck 131 can be any suitable width such as 0.8mm, 0.9mm, 1.0mm, 1.1mm, 1.2mm, or any suitable width within the width range formed by these widths; the width of the extension 133 can be any suitable width such as 3mm, 3.5mm, 3.8mm, 4.0mm, 4.2mm, 4.5mm, 5mm, or any suitable width within the width range formed by these widths.

[0056] In addition, the length of the neck 131 can be 4 to 6 mm (e.g., any suitable length such as 4 mm, 4.5 mm, 5 mm, 5.5 mm, 6 mm, or any suitable length within the length range formed by these lengths), and the height is the same as the height of the extension 133.

[0057] In this embodiment, the extension section 133 is the core capture section of the microfluidic channel 130. Its cross-section can be a standard rectangle with no cross-sectional deformation and no uneven wall thickness. The four sides of the rectangle are perpendicular or parallel to the fluid flow direction to ensure uniform distribution of fluid shear stress in the channel.

[0058] Preferably, the specific dimensions of the extension segment 133 are: width 4 mm, height 250 μm, and length 46.65 mm. P-selectin can be uniformly distributed on the inner wall of the extension segment. Using a height of 250 μm as a baseline, the length:width:height ratio of the extension segment 133 is 186.6:16:1. This size provides sufficient adhesion contact area between the cells / spheres and the P-selectin, while maintaining stable physiological shear stress through the 4 mm × 250 μm cross-sectional dimensions, avoiding low capture efficiency due to excessively short length or shear stress imbalance due to excessively large cross-section.

[0059] The extension section 132 can specifically be a conical extension section with an inverted trapezoidal structure, integrally connecting the neck 131 and the extension section 133. Its small-diameter end is the same width and height as the neck 131 (1mm×250μm), and its large-diameter end is the same width and height as the extension section 133 (4mm×250μm). The upper and lower bottom surfaces are parallel and perpendicular to the fluid flow direction. Both sidewalls of the conical extension section are inclined planes. The angle formed by a single sidewall and the fluid flow direction of the extension section 133 can preferably be 53°. The 53° inclination angle ensures that when the fluid expands from the 1mm wide neck 131 to the 4mm wide extension section 133, the flow path is smooth without abrupt changes, avoiding an increase in the overall channel length due to an excessively small angle (too slow expansion) or an excessively large angle (too rapid expansion) which could lead to fluid turbulence and bubble formation.

[0060] The neck 131 is the guide section of the microfluidic channel 130. Its cross-section is a rectangle with the same height as the extension section 133. The width of the neck 131 can be 1 mm, and the width of the extension section 133 can be 4 mm. The width ratio of the neck 131 to the extension section 133 is 1:4. This ratio is adapted to the 53° included angle of the conical extension section, ensuring that the expansion process of the fluid in the conical extension section is uniform and stable. The narrow width of the guide section enables the concentrated flow of the fluid, and the width of the capture section enables the full adhesion of cells. The two work together to improve the separation efficiency.

[0061] In one embodiment, the radius of curvature of the transition arc between the reservoir 140 and the outlet end of the microfluidic channel 130 is 1.5 mm. Designed based on the 3 mm × 200 μm cross-sectional dimensions of the extension section and a low flow rate (0.05~0.2 μL / s), it adapts to the fluid flow characteristics of small cells, avoiding cell retention due to an excessively large radius of curvature. The extension section 133 has a rectangular cross-section with a width of 3 mm, a height of 200 μm, and a length of 40 mm. Its narrow cross-section and short length meet the adhesion requirements of small cells, avoiding non-specific adhesion. The angle between the single sidewall of the conical extension section and the fluid flow direction is 45°. The small-diameter end connects to the 0.8 mm wide neck 131, and the large-diameter end connects to the 3 mm wide extension section 133. This gentle 45° angle allows for smooth expansion of small cells, avoiding cell impact damage caused by sudden flow rate changes. The neck 131 is 0.8 mm wide, and the extension section 133 is 3 mm wide, ensuring the synergistic effect of cell guidance and adhesion.

[0062] In one embodiment, the radius of curvature of the transition arc between the reservoir 140 and the outlet end of the microfluidic channel 130 is 2.5 mm. Designed based on the 5 mm × 300 μm cross-sectional dimensions of the extension section and a moderate flow velocity (0.2~0.6 μL / s), this design increases the flow space of the curved surface, preventing large spheres from getting stuck at the connection and ensuring turbulent flow. The extension section 133 has a rectangular cross-section with a width of 5 mm, a height of 300 μm, and a length of 50 mm. This wide cross-section and moderate length provide ample space for the flow and adhesion of large spheres. To prevent the sphere from being squeezed and deformed; the angle between the side wall of the conical extension section and the direction of fluid flow is 60°. The small diameter end connects to the 1.2mm wide neck 131, and the large diameter end connects to the 5mm wide extension section 133. The steep 60° angle enables the large-sized sphere to expand rapidly, shortens the length of the expansion section, and prevents the sphere from getting stuck in the expansion section; the neck 131 is 1.2mm wide and the extension section 133 is 5mm wide, increasing the flow space of the neck 131, meeting the flow requirements of the large-sized sphere, and preventing the sphere from getting stuck in the neck 131.

[0063] In one embodiment, combined Figure 3 As shown, a cell separation system is also provided, the system including at least one microfluidic chip as described in any embodiment of this application, and a pressure control component sealed to the microfluidic chip. The pressure control component includes an injection pump and a pressure transmission line. A first end of the pressure transmission line is sealed to the pressure control port of the reservoir 140, and a second end opposite to the first end is connected to the injection pump. A pressure controller 230 is disposed within the pressure transmission line.

[0064] In this embodiment, the cell separation system refers to an integrated cell separation device comprising one or more microfluidic chips as the core separation unit and an integrated pneumatic control component. The pneumatic control component provides precise pneumatic drive to the microfluidic chips, achieving automated, high-throughput, and low-loss separation of target cells. The pneumatic control component is a core functional component that provides precise pneumatic regulation and transmission to the microfluidic chips. It includes an injection pump, pneumatic transmission tubing, and a pneumatic controller 230. By generating a stable positive / negative pressure difference, it drives the directional flow of fluid within the microfluidic chip, replacing the traditional direct fluid drive method, and is key to achieving low-shear stress cell separation. Compared to direct liquid pumping, the pneumatic control component provides smoother control, less pulsation, and reduces the risk of cell shear stress, while also supporting multiplexing. This avoids pressure spikes, uneven extraction, and prolonged cell residence time in the tubing or syringe that can occur with traditional fluid-driven syringe control.

[0065] The pneumatic transmission pipeline refers to the fluid pipeline connecting the syringe pump and the pneumatic control port of the microfluidic chip. It is used to achieve sealed and stable pneumatic transmission. Its structure is adapted to the number of microfluidic chips, enabling a single pump to synchronously drive the pneumatic pressure of multiple chips. It is the core structure for system integration and high throughput. The staged confluence structure refers to the pipeline layout in which multiple branches of the pneumatic transmission pipeline converge to a single main line in sequence. Adjacent sub-segments 240 first merge in pairs to form a first-level confluence segment. Each level of confluence segment 250 then merges upwards in sequence, finally converging at the second end connected to the syringe pump. This ensures the uniform distribution of pneumatic pressure in each branch, achieving synchronous and pressure-equal drive of multiple chips.

[0066] The air pressure controller 230 refers to the air pressure detection and control element configured in the air pressure transmission pipeline. It can detect the air pressure value in the pipeline in real time and adjust the air pressure according to the set value to ensure the stability and uniformity of air pressure transmission and avoid fluid flow rate changes caused by air pressure fluctuations.

[0067] The Luer male connector at the first end of the pneumatic transmission line is snapped shut with the Luer female connector at the microfluidic chip reservoir 140 to prevent pneumatic leakage; the second end of the pneumatic transmission line is threaded and sealed to the pneumatic output port of the precision injection pump, and is fitted with a polytetrafluoroethylene sealing gasket to ensure airtightness under high pressure; the pneumatic controller 230 and the pneumatic transmission line are heat-fused sealed together without any gaps to prevent pneumatic leakage.

[0068] The cell separation system of this application works as follows: A target gas pressure (e.g., 0.03 MPa) is set via an injection pump, and a gas pressure controller 230 detects and provides feedback in real time, stabilizing the gas pressure within the pipeline at the set value. A certain volume (e.g., 100 μL) of suspension containing target cells is injected into the inlet of the microfluidic chip's inlet channel 120 through a P200 pipette tip. The injection pump is then turned on, applying a stable negative pressure to the microfluidic chip's reservoir 140 via the gas pressure transmission pipeline, creating a gas flow between the inlet channel 120 and the reservoir 140. The pressure difference drives the cell suspension to flow directionally and at a constant speed along the inlet channel 120 → microfluidic channel 130 → reservoir 140. The cell suspension flows in a stable laminar flow within the extension 133 of the microfluidic channel 130. Target cells are captured by specific adhesion of P-selectin to the inner wall of the channel, while non-target cells flow into the reservoir 140 with the fluid to complete collection. After separation, the injection pump is turned off, the negative pressure is released, and non-target cells are collected from the reservoir 140. Target cells in the microfluidic channel 130 are then eluted with EDTA buffer to complete the separation.

[0069] The separation system in this application has a simple structure and standardized operation. It achieves precise control of fluid flow rate through closed-loop gas pressure regulation. The separation process does not require manual operation, thus avoiding human error. The negative pressure drive method eliminates the direct contact between mechanical parts and cell suspension, significantly reducing shear stress damage.

[0070] In one embodiment, the microfluidic chip comprises N, where N is a positive integer greater than or equal to 2; the number of first ends of the pneumatic transmission pipeline is consistent with the number of microfluidic chips, and each first end is sealed and connected to each microfluidic chip; the pneumatic transmission pipeline has a step-by-step converging structure, including sub-segments 240 connected to each first end, multiple sub-segments 240 converging to form a first-level converging segment, and each level of converging segment 250 converging upwards step by step, finally converging to the second end; each sub-segment 240 and each converging segment 250 is equipped with a pneumatic controller 230.

[0071] In this application, N can be any suitable positive integer from 2 to 10, and it can be a base number or an even number, such as any suitable even number value like 2, 4, 6, 8, or 10. The microfluidic chips can be arranged in an array, all sealed to a cascading pneumatic transmission pipeline of a pneumatic control component, and driven by a single syringe pump to provide synchronous, uniform negative pressure, enabling parallel and synchronous separation of multiple samples. Each sub-segment is equipped with one or more pneumatic controllers, each controller controlling the pneumatic pressure of its respective sub-segment to achieve extraction of samples from specific microfluidic chips.

[0072] In this embodiment, Figure 3 For example, with N=4, the step-by-step merging pneumatic transmission pipeline is a two-stage step-by-step merging structure of "4→2→1". The system consists of four sub-segments. The first end of each sub-segment is a standard Luer male connector, which snaps into and seals with the corresponding Luer female connector of a microfluidic chip. The four sub-segments are arranged symmetrically, each connecting to one of the four microfluidic chips. There are two primary confluence segments, each formed by the smooth arc convergence of two adjacent sub-segments at the same horizontal height. The confluence point is a smooth arc transition with a suitable radius of curvature (e.g., 3mm), without sharp angles or throttling. The inner diameter of the primary confluence segment is 1.2mm, and the outer diameter is 3.0mm, larger than the inner diameter of the sub-segments to ensure lossless air pressure convergence. There is one secondary confluence segment, formed by the smooth arc convergence of two primary confluence segments at the same vertical height. The confluence point is greater than or equal to the inner diameter of the primary confluence segment, for example, a radius of curvature of 4mm at the confluence. The inner diameter of the secondary confluence segment is 1.5mm, and the outer diameter is 3.5mm. The end of the secondary confluence segment furthest from the primary confluence segment is the second end of the pipeline, which is threaded and sealed to the air pressure output port of the precision injection pump. All junctions of the pipeline have smooth, rounded transitions, with no sudden changes in cross-section or throttling structures, ensuring uniform distribution of air pressure during the merging process.

[0073] There are a total of 6 distributed air pressure controllers, with one in each of the 4 sub-segments and 3 merging segments. They monitor the air pressure values ​​in the 4 sub-segments and 3 merging segments in real time and synchronously feed back all the detection signals to the precision injection pump. The injection pump automatically adjusts the air pressure output according to the feedback signal to achieve closed-loop control of distributed air pressure throughout the pipeline, ensuring that the air pressure in the 4 sub-segments is completely uniform and avoiding deviations in separation results caused by air pressure differences between channels.

[0074] The separation system in this embodiment realizes an integrated design of one pump controlling multiple chips, and the multi-channel parallel separation greatly improves the separation efficiency; the step-by-step confluence pipeline + distributed pressure controller ensures the uniformity of pressure in each channel, which is suitable for batch testing of clinical samples.

[0075] In one embodiment, a microfluidic chip is also provided for cell separation, or a cell separation system is provided for cell separation, for separating target cells and non-target cells.

[0076] Taking highly metastatic cells / spheres as the target system as an example, a single-chip or multi-chip cell separation system can be used. The target cells can be highly metastatic cells to be separated, such as HEYA8 HM, Kuramochi, and SKOV3 M-CSC, while the non-target cells are low-metastatic cells such as OVSAHO and CaOV3, in order to obtain high-purity target cells and non-target cells.

[0077] Furthermore, this application can also be applied to fields such as anti-tumor drug screening (separating tumor cells after drug treatment and detecting the killing effect of drugs on cells with different metastatic characteristics) and tumor stem cell separation (separating tumor stem cells with high metastatic potential from tumor tissue), adapting to various tumor-related cell separation needs. Compared with traditional cell separation methods (such as density gradient centrifugation and immunomagnetic bead sorting), the microfluidic chip or cell separation system of this application has significant advantages in cell separation applications, such as low sample consumption, fast separation speed, high specificity, low cell damage, and high-throughput operation. Moreover, the chip is miniaturized and can realize rapid on-site detection, which has important application value in the fields of basic tumor research and clinical testing.

[0078] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit them. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of this application.

[0079] Furthermore, those skilled in the art will understand that although some embodiments herein include certain features included in other embodiments but not others, combinations of features from different embodiments are meant to be within the scope of this application and form different embodiments. For example, all the embodiments above can be used in any combination. The information disclosed in this background section is intended only to enhance the understanding of the general background of this application and should not be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

Claims

1. A microfluidic chip for cell separation, characterized in that, include: Chip body; The chip body has a liquid inlet channel, a non-blocking microfluidic channel, and a liquid reservoir connected to the outlet end of the microfluidic channel. The inner wall of the microfluidic channel is modified with P-selectin for capturing target cells. The microfluidic channel is an integrated anti-bubble structure, which includes a neck, an extension section, and an extension section in sequence along the fluid flow direction. The extension section is connected to the liquid storage tank.

2. The microfluidic chip according to claim 1, characterized in that, The liquid inlet channel extends from the upper surface of the chip body into the interior of the chip body and is smoothly connected to the inlet end of the microfluidic channel. The microfluidic channel extends inside the chip body and is smoothly connected to the liquid reservoir at the outlet end. The liquid reservoir extends from inside the chip body out of the upper surface of the chip body.

3. The microfluidic chip according to claim 2, characterized in that, The upper surface of the liquid storage tank is a circular planar structure, which is used to connect the upward-extending Luer connector to form the liquid storage section.

4. The microfluidic chip according to claim 1, characterized in that, The liquid storage tank and the outlet end of the microfluidic channel are connected by an arc transition, and the radius of curvature of the transition arc is adapted to the requirement of non-turbulent fluid flow.

5. The microfluidic chip according to claim 1, characterized in that, The extension section has a rectangular cross-section, and the width of the extension section is 3~5mm, the height is 200~300μm, and the length is 30~80mm; and / or The ratio of the length:width:height of the extension segment is (120~300):(12~30):

1.

6. The microfluidic chip according to claim 1, characterized in that, The extension section is a tapered extension section, and the angle between the edge of the tapered extension section and the fluid flow direction of the extension section is 30°~60°.

7. The microfluidic chip according to claim 6, characterized in that, The width of the neck is 0.8~1.2mm, and the width of the extension is 3~5mm.

8. A cell separation system, characterized in that, The system includes at least one microfluidic chip as described in any one of claims 1 to 7, and a pneumatic control component that is hermetically connected to the microfluidic chip; The pneumatic control component includes an injection pump and a pneumatic transmission pipeline. The first end of the pneumatic transmission pipeline is sealed to the upper end face of the liquid storage tank, and the second end opposite to the first end is connected to the injection pump. A pneumatic controller is configured inside the pneumatic transmission pipeline.

9. The cell separation system according to claim 8, characterized in that, The microfluidic chip comprises N chips, where N is a positive integer greater than or equal to 2; The number of first ends of the air pressure transmission pipeline is the same as the number of microfluidic chips, and each first end is sealed and connected to each microfluidic chip in a one-to-one correspondence. The pneumatic transmission pipeline has a step-by-step converging structure, including sub-segments connected to each first end. Multiple sub-segments merge to form a first-level converging segment, and each level of converging segment merges upwards in sequence, finally converging to the second end. Each sub-segment and merging segment is equipped with a pressure controller.

10. The application of the microfluidic chip as described in any one of claims 1 to 7 or the cell separation system as described in any one of claims 8 to 9 in cell separation, characterized in that, Used for separating target cells from non-target cells.

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