Cell communication micro-fluidic chip with cell capture structure and application thereof

Abstract

The invention relates to the technical field of microfluidics, and particularly discloses a cell communication micro-fluidic chip with a cell capturing structure and application of the cell communication micro-fluidic chip. The chip comprises main channels, and the two main channels are arranged in parallel at an interval; the cell interception structures are arranged in the main channels, grooves capable of accommodating at least one cell to be cultured are formed in the incident flow surfaces in the main channels, and at least one opening only allowing a culture solution to flow out is formed in each groove, so that the cells can stably stay and are uniformly distributed, and meanwhile, fluid is allowed to pass through; the culture solution interaction structure is arranged between the two main channels and used for interaction of culture solutions in the two main channels, transverse exchange of the culture solutions and cell secretions is achieved, cell migration is prevented at the same time, and therefore the non-contact cell communication model is constructed. The cell communication micro-fluidic chip can efficiently simulate the cell paracrine communication process, and is suitable for various cell communication research scenes such as inflammatory response, immune regulation, tumor microenvironment, stem cell secretion factor effect and the like.

Description

Technical Field

[0001] This invention relates to the field of microfluidics, specifically to a cell communication microfluidic chip with a cell capture structure and its applications. Background Technology

[0002] Intercellular communication plays a crucial role in immune regulation, tissue repair, inflammatory responses, neural signal transduction, and tumor metastasis. Different cell types typically exchange substances and signals by secreting cytokines, exosomes, or metabolites. In-depth research into these processes is of great significance for elucidating disease mechanisms and developing novel therapeutic approaches.

[0003] Organoids are three-dimensional micro-organ models formed by culturing stem cells or tissue cells in vitro. Their structure, cell types, and functions closely mimic real organs, providing a more physiologically accurate experimental platform for scientific research. By constructing interaction systems between organoids and other cell types (such as immune cells and nerve cells), it is possible to dynamically simulate the complex intercellular communication mechanisms involved in organ development, homeostasis maintenance, and disease development, including paracrine signal transduction and cell-contact-dependent signal regulation. This model has significant research value for a deeper understanding of life processes and disease mechanisms.

[0004] Traditional cell communication research often relies on methods such as Transwell intercalation, conditioned medium collection and reprocessing, or micropipette manipulation. However, these methods have many limitations, such as difficulty in dynamically observing cell-cell interactions, uncertainty in factor diffusion pathways, unstable cell states, poor reproducibility of results, and difficulty in precisely controlling the experimental system in terms of time and space.

[0005] Microfluidics, with its microscale control capabilities and highly integrated characteristics, has been increasingly applied to cell culture and communication research. In recent years, researchers have developed various microfluidic co-culture chips, employing parallel channels, isolating membranes, porous microgrooves, or gradient-forming structures to achieve intercellular fluid exchange or factor delivery. Compared to traditional methods, these devices significantly improve experimental efficiency and controllability, demonstrating great potential in tissue microarrays, tumor microenvironment simulation, and immune cell activation.

[0006] However, existing microfluidic chips still face several technical challenges in cell communication applications: (1) Cells are difficult to stay stably in the channel and are easily washed away by the fluid, resulting in uneven adhesion or loss; (2) Some designs rely on membranes or fixed channels to highly block cells, and the manufacturing process is complex and has poor adjustability; (3) Under different experimental requirements, the chip structure lacks compatibility with different cell types and sizes, and has poor adaptability; (4) Limited liquid exchange efficiency may affect the diffusion of communication factors and the authenticity of reaction results.

[0007] Therefore, there is an urgent need for a cell communication microfluidic chip with a reasonable structure, high liquid exchange efficiency, good cell interception effect, and certain adaptability and adjustability to meet the experimental needs of research on non-contact communication mechanisms between various types of cells. Summary of the Invention

[0008] The purpose of this invention is to address the aforementioned shortcomings of the prior art by providing a cell communication microfluidic chip with a cell capture structure and its application. This chip enables non-contact co-culture of different types of cells, supports efficient liquid factor exchange, and has advantages such as adjustable structure, controllable experiment, and simple manufacturing process.

[0009] To achieve the above objectives, the present invention adopts the following technical solution: The first objective of this invention is to provide a cell communication microfluidic chip with a cell capture structure, comprising: The main channel consists of two parallel main channels spaced apart. One end of each main channel is configured as the first liquid inlet, and the other end is configured as the first liquid outlet. A cell interception structure is disposed within each main channel. The cell interception structure has a groove on the flow-facing surface within the main channel capable of accommodating at least one cultured cell. The groove has at least one opening for culture medium outflow only. The culture medium exchange structure is located between the two main channels to allow for the exchange of culture medium between the two main channels.

[0010] Furthermore, the cell interception structures are arranged in rows along the flow direction of the culture medium in the main channel, with at least one cell interception structure in each row. The cell interception structures in adjacent rows are staggered, and the gap between two cell interception structures in the upstream row is directly opposite to the cell interception structure in the adjacent downstream row.

[0011] Furthermore, the cell interception structure is formed by two L-shaped blocking structures with a gap between them, a groove is formed between the two arms of the two L-shaped blocking structures along the flow direction, and an opening is formed between the two arms of the two L-shaped blocking structures perpendicular to the flow direction.

[0012] Furthermore, the culture medium interaction structure is a grid-like microchannel that directly connects the two main channels. The grid-like microchannel is composed of a number of microgrooves arranged at intervals along the fluid flow direction, and the size of the microgrooves is configured to allow only the culture medium to pass through.

[0013] Furthermore, the culture medium interaction structure includes a grid-like microchannel connected to the opposite side of each main channel and a cell culture communication layer connecting two grid-like microchannels.

[0014] Furthermore, the cell culture communication layer includes a group of culture chambers connected to the main channel via a grid-like microchannel and a central exchange chamber connected to the two groups of culture chambers via branch channels.

[0015] Furthermore, the culture chambers in the culture chamber group are connected in series, and each culture chamber is connected to the corresponding main channel through a grid-like microchannel.

[0016] Furthermore, the first and last culture chambers in the culture chamber group are respectively provided with a second liquid inlet and a second liquid outlet.

[0017] The second objective of this invention is to provide the application of the above-mentioned cell communication microfluidic chip in non-contact cell co-culture for the study of cell communication mediated by secretory factors between different cell lines.

[0018] Furthermore, it is also applied to biological research related to cell communication. The cell communication microfluidic chip is used to realize the in vitro simulation and detection of intercellular signal transduction, secretory factor exchange and microenvironment interaction, including inflammatory response, immune response, tumor microenvironment regulation, drug screening and stem cell secretory factor function analysis.

[0019] Compared with the prior art, the beneficial effects of the technical solution provided by the present invention are as follows: (1) Non-contact communication simulates reality: Liquid exchange is achieved through microchannels instead of direct cell contact, which better simulates the paracrine communication process in vivo; (2) Cell stability: The unique interception structure allows cells to be distributed in an orderly manner within the main channel, reducing drift and loss; (3) High-efficiency liquid exchange: The microchannel design ensures rapid lateral diffusion of signaling factors, improving the efficiency of communication reactions; a "flow resistance buffer zone" is formed between the main channel and the cell growth zone, allowing fresh culture medium to enter the adhesion zone through diffusion rather than direct rinsing. This ensures sufficient nutrient renewal and effectively prevents the erosion and shedding of early-adhesive cells caused by high flow rates.

[0020] (4) High adaptability: The channel structure and interception structure are adjustable, suitable for various cell types and experimental needs; (5) Easy to manufacture and integrate: simple structure, can be mass-produced, and easy to use with conventional experimental platforms (microscopes, petri dishes); (6) Strong verification: This chip has been successfully applied in cytokine-induced inflammation experiments (such as Hep G2 and U87 cells), verifying the effectiveness and practicality of its structural design. Attached Figure Description

[0021] Figure 1This is a schematic diagram of the structure of a cell communication microfluidic chip according to the present invention; Figure 2 This is an exploded view of the structure of the first version of the cell communication microfluidic chip of the present invention without a cell interception structure. In the figure, A is the design diagram; B is an enlarged view of the main channel design, in which the main channel width is 500 μm and the microgroove length is 300 μm; C is an enlarged view of the cell interception structure and microgroove design, in which the microgroove width is 10 μm. Figure 3 The images show actual micrographs of the first version of the cell communication microfluidic chip of the present invention without a cell interception structure. In the figure, A is a 4x optical micrograph of the actual chip, and B is a 10x optical micrograph of the main channel portion of the chip. Figure 4 This is an exploded view of the structure of a cell communication microfluidic chip with a cell interception structure in the second version of the present invention. In the figure, A is the design diagram; B is an enlarged view of the main channel design, in which the microgroove length is 400 μm, the main channel width is 400 μm, and the cell interception structure is arranged in a 1-2-1-2 array with gap widths of 40 μm and 130 μm, respectively; C is an enlarged view of the cell interception structure and microgroove design, in which the opening width inside the groove of the cell interception structure is 10 μm, and the microgroove width is 10 μm. Figure 5 This is an enlarged schematic diagram of the microchannel structure of the cell communication microfluidic chip with a cell interception structure in the second version of the present invention. Figure 6 The images show actual micrographs of the cell communication microfluidic chip with a cell interception structure in the second version of the present invention. In the figure, A is a 4x optical micrograph of the actual chip, and B is a 10x optical micrograph of the main channel portion of the chip. Figure 7 This is an exploded view of the structure of a cell communication microfluidic chip with a cell interception structure in the third version of the present invention. In the figure, A is the design diagram; B is an enlarged view of the main channel design, in which the microgroove length is 200 μm, the main channel width is 400 μm, and the cell interception structure is arranged in a two- or three-array combination, with gap widths of 55 μm and 25 μm respectively; C is an enlarged view of the cell interception structure and microgroove design, in which the internal opening width of the U-shaped structure of the cell interception structure is 5 μm, and the microgroove width is 5 μm. Figure 8 This is an enlarged schematic diagram of the microchannel structure of a cell communication microfluidic chip with a cell interception structure, as shown in the third version of the present invention. Figure 9 The images show actual micrographs of the final version of a cell communication microfluidic chip with a cell capture structure according to the present invention. In the figure, A is a 4x optical micrograph of the actual chip, and B is a 10x optical micrograph of the main channel portion of the chip. Figure 10 A schematic diagram of a cell communication microfluidic chip with a cell capture structure; Figure 11 Photograph of a microfluidic chip being filled with ink; Figure 12 Bright field and immunofluorescence images of the microfluidic chip of the present invention characterizing the effect of HepG2 cells on U87 cells are shown in the figure. A is the bright field image and B is the immunofluorescence image. Figure 13 This is a schematic diagram of the structure of a non-contact cell co-culture device provided by the present invention; Figure 14 A schematic diagram of organoid-cell interactions; Figure 15 The image shows a physical diagram of a non-contact co-culture device for cells provided by the present invention. The left image is a photograph of the PDMS bilayer chip after alignment, and the right image is a microscopic diagram of the interaction channels of the PDMS bilayer chip. Figure 16 The images show the early stages of organoid culture. The left image is a 4x magnification image of the early stages of organoid culture in a microarray, and the right image is a 10x magnification image of the early stages of organoid culture in a microarray.

[0022] In the picture: 100 - Main channel, 110 - First inlet, 120 - First outlet; 200 - Cell interception structure, 210 - Groove, 211 - L-shaped barrier structure, 220 - Opening; 300 - Culture medium interaction structure, 310 - Palletized microchannel, 311 - Microgroove; 400 - Cell culture communication layer, 410 - Culture chamber group, 4101 - First culture chamber group, 4102 - Second culture chamber group, 411 - Culture chamber, 412 - Second liquid inlet, 413 - Second liquid outlet, 420 - Branch channel, 430 - Central exchange chamber; 510 - Substrate, 520 - Cover plate. Detailed Implementation

[0023] To make the objectives, technical solutions, and advantages of this invention clearer, the specific embodiments and accompanying drawings are described in further detail below. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0024] In the description of this invention, it should be noted that the terms "center," "longitudinal," "lateral," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," and "outer," etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are used only for the convenience of describing the invention and for simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on the invention. Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance.

[0025] In the description of this invention, it should be noted that, unless otherwise explicitly specified and limited, the terms "connected" and "linked" 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. Those skilled in the art can understand the specific meaning of the above terms in this invention based on the specific circumstances.

[0026] Example 1 like Figure 1 As shown, this embodiment provides a cell communication microfluidic chip with a cell capture structure, including: The main channel 100 consists of two parallel main channels spaced apart. Each main channel has a first inlet at one end and a first outlet at the other end. These channels are responsible for the input and delivery of cell suspension and the continuous perfusion of nutrients and medications during subsequent culture. A cell interception structure 200, which uses physical confinement to precisely capture and fix cells, is disposed within each main channel. The cell interception structure has a groove on the flow-facing surface within the main channel capable of accommodating at least one cell to be cultured, and the groove has at least one opening for culture medium outflow only; and The culture medium interaction structure 300 is located between the two main channels to facilitate the exchange of culture medium within the two main channels. It achieves efficient diffusion and exchange of nutrients and metabolites through tiny gaps, while protecting cells from mechanical shearing damage caused by high flow rates.

[0027] The main channel 100 serves as the feed channel for the cell suspension and is connected to the flow path of the cell interception structure 200, enabling efficient cell loading. The cell interception structure 200 uses preset geometric dimensions to physically confine the incoming cells, ensuring they enter a confined area within the structure along with the fluid. Due to the flow resistance difference between this area and the main channel, a localized low-velocity zone is formed, promoting gravitational sedimentation of cells on the substrate surface surrounded by the cell interception structure. Combined with an adhesion-promoting coating on the substrate surface, cells achieve targeted adherence and growth in this area. Therefore, in the early stages of adherent culture, the cell interception structure 200 can guide cells to settle uniformly within a specific culture chamber, ensuring a consistent spatial distribution of adherent cells on the chip and providing a standardized cell base for subsequent high-throughput screening.

[0028] According to fluid dynamics simulations, the culture medium interaction structure 300, namely the groove 210 connecting the main channel 100 and the cell interception structure, has a significant flow resistance regulation function. When the main channel 100 is subjected to high-speed perfusion to replace the culture medium, most of the fluid is confined within the main channel, while the fluid velocity entering the cell adhesion zone is significantly reduced, forming a static microenvironment. This effectively avoids cell detachment, drift, or morphological abnormalities due to excessive mechanical shear forces in the early stages of adhesion, ensuring the stability of adherent cell function.

[0029] Utilizing the principle of diffusion, fresh nutrients, dissolved oxygen, and test drugs in the main channel continuously migrate to the cell interception zone through the culture medium interaction structure. At the same time, cell metabolites diffuse backward into the main channel and are carried away. This exchange method not only ensures the long-term activity of adherent cells, but also creates an extremely stable concentration gradient in the interception zone when different component liquids are introduced into the main channels on both sides.

[0030] Because the cell-intercepting structure locks adherent cells in a fixed area, the microscopic optical system can achieve multi-site, long-term in-situ automated imaging. Moreover, the adherent cells form tiny tissue sheets within the specific structure, facilitating in-situ fixation, staining, or single-cell sampling after the experiment, thus improving the accuracy of the analysis.

[0031] In some embodiments, the cell interception structures 200 are arranged row by row along the flow direction of the culture medium within the main channel 100, with at least one cell interception structure in each row. Adjacent rows of cell interception structures are staggered, with the gap between two cell interception structures in the upstream row aligned with the adjacent cell interception structure in the downstream row. This staggered arrangement eliminates straight-line through-flow channels, altering the trajectory of cells within the main channel. This ensures that uncaptured cells from the upstream row collide with and enter the effective capture zone of the downstream interception structure after passing through the gap, achieving layer-by-layer filtration of cells in the fluid, minimizing cell loss, and maximizing the capture rate. The design of the upstream gap aligned with the downstream interception structure generates subtle disturbances when the fluid collides with the interception wall. This dynamic effect promotes the diffusion rate of nutrients from the main channel into the interception structure, enhancing material exchange efficiency while maintaining low shear force. It effectively balances the flow resistance throughout the chip, ensuring consistency in large-scale adherent culture. Furthermore, it provides maximized effective adhesion sites within a limited interception area, increasing experimental throughput.

[0032] In some implementations, the number of cell interception structures 200 included in the uplink is N, and the number of cell interception structures 200 included in the corresponding downlink is M, and N = M + 1, where M is an integer greater than or equal to 1.

[0033] More specifically, when M=1, the cell interception structure 200 exhibits an alternating arrangement of 2-1-2-1-2; when M=2, the cell interception structure 200 exhibits an alternating arrangement of 3-2-3-2-3.

[0034] like Figure 1 As shown, the cell interception structure 200 is arranged in a 2-1-2-1-2 pattern within the main channel 100.

[0035] In some embodiments, the cell interception structure 200 is formed by two L-shaped blocking structures 211 spaced apart. A groove 210 is formed between the two arms of the two L-shaped blocking structures 211 along the flow direction, and an opening 220 is formed between the two arms of the two L-shaped blocking structures 211 perpendicular to the flow direction. By using a concave cell interception structure, the fluid resistance at the opening end guides the cells to settle precisely at the bottom of the groove, significantly reducing random cell drift and loss.

[0036] In some embodiments, the culture medium interaction structure 300 is a grid-like microchannel 310 that directly connects two main channels. The grid-like microchannel 310 is composed of a plurality of microgrooves 311 arranged at intervals along the fluid flow direction. The microgrooves 311 are sized to allow only the passage of culture cells. While ensuring fluid flow, it strictly prevents cells from crossing into the main channels, ensuring that the positioning and distribution of the adherent cell population are not disrupted.

[0037] In some embodiments, to accommodate cell types of different sizes, the opening 220 of the cell interception structure has a width of 2-10 micrometers, and the microgroove 311 has a width of 2-10 micrometers, to prevent cell passage and ensure smooth fluid exchange. This is applicable to adherent cells with a diameter of 8-20 micrometers, such as immune cells, stem cells, epithelial cells, and tumor cells.

[0038] In some embodiments, the culture medium interaction structure 300 includes a grid-like microchannel 310 connected to the opposite side of each main channel and a cell culture communication layer 400 connecting two grid-like microchannels.

[0039] In some embodiments, the structure of the cell culture communication layer 400 is not limited, and it includes a culture chamber group 410 connected to the main channel through a grid-like microchannel and a central exchange chamber 430 connected to the two culture chamber groups through branch channels 420 respectively.

[0040] refer to Figure 13 The cell culture communication layer 400 is centered on the central exchange chamber 430 and is branched by the branch channels 420 into a first culture chamber group 4101 and a second culture chamber group 4102. The first culture chamber group 4101 and the second culture chamber group 4102 are each connected in series by multiple culture chambers 411. The first culture chamber group 4101 and the second culture chamber group 4102 are each provided with a second liquid inlet 412 and a second liquid outlet 413 to facilitate liquid exchange. The bottom edge of the culture chamber 411 is connected to the grid-like microchannel 310 to realize liquid exchange between channels and prevent cells from passing through.

[0041] It should be noted that the structure of the branch channel 420 is not limited. To facilitate cell cluster formation in the culture chamber, the branch channel 420 can be designed as a dendritic channel in this embodiment. Through multi-level symmetrical branching, the liquid in the main channel is proportionally and progressively distributed to each terminal capture zone. While ensuring high-throughput liquid exchange, the flow velocity at the branch ends is naturally reduced to an extremely low level. This provides an ultra-low shear stress growth environment for adherent cells, greatly improving the cell adhesion success rate and long-term survival rate, and more realistically simulating the human physiological environment.

[0042] The structure of the culture chamber group 410 is not limited. In order to facilitate the formation of cell clusters in the culture chamber, the culture chamber 411 in this embodiment can be designed as a cone-shaped structure with an upward protrusion in each culture chamber. After molding, it becomes a downward concave cone-shaped bottom, which facilitates the formation of cell clusters in the cone-shaped bottom chamber. The edge of the cone-shaped bottom of the culture chamber is connected to the main channel 100 through a grid-like microchannel 310 to realize the liquid exchange between the two channels and at the same time prevent cells from passing through.

[0043] In some embodiments, the fluid-driven method is injection pump-driven, liquid level difference-driven, pressure difference-driven, or gravity-driven. Specifically, the cell loading method is as follows: the cell suspension is added to the inlet end, and the cells are aspirated from the outlet end by the injection pump at a rate of 0.01~0.05 mL / min, so that the cells are gradually captured and fixed by the structure.

[0044] It should also be noted that, as Figure 10 As shown, the main channel, cell interception structure, and culture medium interaction structure in this invention are all formed by processing channels on the substrate 510 and covering them with a cover plate 520. Therefore, the channel size generally refers to the channel width, and the channel height H is not limited. Under normal circumstances, in order to ensure that all cells can pass through, the channel height should be greater than the diameter of the largest cell.

[0045] Example 2 To obtain a stable and controllable cell communication microfluidic chip, this invention underwent multiple structural optimizations: The first design: The chip consists of two main channels and a central microgroove. Both the main channels and the central microgroove are relatively wide, and no cellular interception structure is included. Design drawings and physical prototypes are shown below. Figure 2 , Figure 3 As shown in the figure. In the cell loading experiment, it was found that the cells could not remain in the main channel due to the fluid flow, making it difficult to form a stable culture system.

[0046] Second design: A cell interception structure was added to the main channel to achieve cell retention. Design drawings and physical prototype images are shown below. Figure 4 , Figure 5 , Figure 6 As shown. However, due to the use of film mask processing, the minimum width of the opening of the cell interception knot and the middle microgroove can only be controlled within 10 μm. In practical applications, this size failed to effectively prevent cells from passing through, resulting in cell mixing in the main channels on both sides and unsatisfactory communication isolation.

[0047] The third optimized design employs a chrome-plated glass mask process, adjusting the width of the openings and central microgrooves of the cell interception structure to 2-10 μm, and increasing the density and number of the cell interception structure and central microgrooves. Design drawings and physical prototype images are shown below. Figure 7 , Figure 8 , Figure 9 As shown, the optimized chip effectively intercepts cells, ensuring their stable attachment to the main channel. Simultaneously, the microgrooves facilitate the exchange of liquid factors and prevent cell transgression, achieving ideal experimental results.

[0048] The above iterative process shows that, through refined structural design, the present invention has successfully solved the problems of cell retention and insufficient isolation in the prior art, providing a reliable chip platform for the final realization of stable non-contact cell co-culture.

[0049] Example 3 This embodiment provides a microfluidic chip, which includes a cover plate and a substrate. The cover plate is disposed on the substrate, and a cell communication microfluidic chip with a cell capture structure is disposed on the cover plate. The provided microfluidic chip is as follows: Figures 7-9 As shown.

[0050] The fabrication method of the microfluidic chip in this embodiment includes the following steps: (1) Mask design The pattern of a cell communication microfluidic chip with a cell capture structure was drawn using AutoCAD drawing software. The pattern was then transferred to a chrome-plated glass plate, and then transferred to a photoresist under ultraviolet light.

[0051] The dimensions of the cell communication microfluidic chip with cell trapping structure are as follows: the length of the first main channel and the second main channel are 4400 μm, the width is 400 μm, and the height is 25 μm; the cell interception structure is set in the middle of the main channel and arranged in an array of opening structures, with the bottom opening width of each structure being approximately 5 μm; the microgroove structure is set between the two main channels, with each microgroove being approximately 5 μm wide and 200 μm long.

[0052] (2) Preparation of PDMS thin film The SU-8 positive mold was fabricated using soft photolithography. SU-8 photoresist (3025) was evenly spread on a clean and dried silicon wafer (800 rpm for 40 s, 3000 rpm for 60 s), and then preheated on a hot plate to remove the solvent from the photoresist (65℃ for 10 min, 90℃ for 25 min). Photolithography was then performed (6 s, 5 mJ / cm²). 2 The material is then placed on a hot plate for post-baking (65 ℃ for 2 min, 90 ℃ for 8 min). After PGMEA development and isopropanol fixing, it is then molded on a hot plate (135 ℃ for 120 min) to obtain a positive mold with a microstructure. After creating the positive mold, its structure is copied onto a PDMS sheet using a rapid prototyping method. Specifically, PDMS is mixed with a curing agent in a 10:1 ratio, and the gas is removed to obtain a precursor. This precursor is poured onto the positive mold and heated in an oven at 75 ℃ for 2 h. The cured PDMS is then peeled off to obtain a PDMS sheet with a thickness of approximately 3 mm. Figure 9 The image shown is a micrograph of a cell communication chip on a PDMS sheet.

[0053] (3) PDMS thin layer is bonded to glass substrate to form microfluidic chip Perforations were made at the inlet and outlet using manual punches of 3 mm and 1 mm diameter, respectively. The PDMS sheet was cleaned with alcohol and dried under nitrogen to ensure no debris was removed. The structured side of the PDMS layer was then attached to the bottom of a 35 mm cell culture dish. Finally, a cylindrical reservoir with a diameter of 6 mm and a height of 6 mm was attached to the inlet using AB glue. The dish was then heated at 65°C for 2 hours to ensure a tight bond between the reservoir and the PDMS layer. An image of the microfluidic chip is shown below. Figure 10 and 11 As shown, where Figure 11 Photograph of a cell communication microfluidic chip filled with ink.

[0054] (4) Cell loading and culture To load cells into the chip for culture, the required cell suspension must first be prepared. Connect both ends of a 25 cm silicone tube to a stainless steel tube and a flat-tipped needle, respectively. Clean the tube with alcohol and water, then purge it with nitrogen. Insert the other end of the stainless steel tube into the pre-drilled hole at the outlet of the PDMS layer. Next, add the cell suspension to the reservoir at the chip's inlet. Use a syringe pump to aspirate the cells from the outlet at a rate of 0.01 mL / min, allowing them to enter the channels with the fluid. During the flow, the cells are trapped by the capture structure array and remain in the main channels, gradually attaching and growing. Finally, place the chip in a standard cell culture incubator (37°C, 5% CO2) for further culture.

[0055] Example 4 To verify the effectiveness of the chip of this invention, a non-contact co-culture experiment was conducted using the human hepatocellular carcinoma cell line Hep G2 and the human glioma cell line U87: Hep G2 cells were seeded in the first main channel, and U87 cells were seeded in the second main channel; the cells were co-cultured for 24 hours, and liquid exchange occurred through the microgroove in the middle of the chip; after 24 hours, immunofluorescence staining was performed to detect the expression levels of inflammatory factors (CD86, CD206) in U87 cells. Experimental results are as follows Figure 12 As shown, A is a bright-field image and B is an immunofluorescence image. The results indicate that, compared with single culture, Hep G2 under different modeling conditions in co-culture had different effects on the expression of the above-mentioned inflammatory factors in U87 cells, suggesting that Hep G2 secretions can transmit influencing signals through liquid exchange via microgrooves.

[0056] This experiment demonstrates that the chip of the present invention can effectively simulate non-contact communication mechanisms between different cells, verifying the rationality of the design of the cell interception structure and the culture medium interaction structure.

[0057] Example 5 This embodiment provides a non-contact cell co-culture device.

[0058] refer to Figure 13 It includes: a main channel, two main channels arranged in parallel with a gap between them, one end of each main channel is configured as the first liquid inlet and the other end is configured as the first liquid outlet; A cell interception structure is disposed within each main channel. The cell interception structure has a groove on the flow-facing surface within the main channel capable of accommodating at least one cultured cell. The groove has at least one opening for culture medium outflow only. A culture medium interaction structure, positioned between two main channels, facilitates the exchange of culture medium within the two main channels. This culture medium interaction structure 300 includes a grid-like microchannel 310 connected to the opposite side of each main channel and a cell culture communication layer 400 connecting the two grid-like microchannels. The cell culture communication layer 400 includes a group of culture chambers 410 connected to the main channels via the grid-like microchannels and a central exchange chamber 430 connected to the two culture chamber groups via branch channels 420. More specifically, the cell culture communication layer 400, centered on the central exchange chamber 430, branches vertically and vertically into the culture chamber groups 410. Each culture chamber 411 is interconnected by a second inlet 412 on the left and a second outlet 413 on the right. The conical bottom edge of each culture chamber 411 communicates with the grid-like microchannels 310, enabling fluid exchange between the channels while preventing cell passage.

[0059] refer to Figure 14 This is a schematic diagram illustrating the interaction between organoids and cells. The organoid culture process is shown below: To form organoids in the cone-shaped base of a chip, a cell suspension must first be prepared in advance. A 25 cm silicone tube was connected at both ends to a stainless steel tube and a flat-headed needle, respectively. The tube was cleaned with alcohol and water, then purged with nitrogen. Cell suspension was then drawn using a syringe, and the other end of the stainless steel tube was inserted into the central perforation of the PDMS layer, allowing the suspension to flow into a conical-bottom culture tank. During the flow, the cells branched out along the tree-like structure, converging at the bottom of the cone to grow in suspension. Finally, the chip was placed in a standard cell culture incubator (37°C, 5% CO2) and incubated for 24 hours to form 3D cell spheroids for further culture.

[0060] A physical diagram of the non-contact cell co-culture device constructed in this embodiment is shown below. Figure 15 As shown, the red part in the left image is the organoid culture area, and the blue part is the immune cell culture area; the right image is an interaction channel diagram under a double-layer chip microscope, in which the cell interception structure is arranged in alternating rows of 3-2-3-2-3.

[0061] refer to Figure 16The images show the initial stages of organoid culture. The left image (4x magnification) shows the overall microstructure of the chip. A circular culture chamber with a clear flow channel design is visible. The area within the red box is the core area for cell seeding; due to the high cell density or the presence of scaffold material, it appears as a deep shadow under low magnification. The right image (10x magnification) shows a fine mesh-like or granular texture in the central area, which typically indicates that the initially seeded single cells or tiny cell clusters are evenly distributed within the hydrogel matrix.

[0062] The cell communication microfluidic chip provided in this embodiment offers a superior physiological model for simulating the complex interactions between organoids and immune cells, thanks to its precise microstructure design. Its central circular microcavity utilizes a hydrogel matrix to construct a highly biomimetic three-dimensional space, inducing polarized tissue structures within the organoid. Simultaneously, interconnected microfluidic channels on both sides simulate the in vivo circulatory system, enabling immune cells (such as T lymphocytes or NK cells) to migrate directionally under a preset flow pressure. This design reproduces the dynamic process of immune cell recruitment, penetration, and destruction of tumors or tissue lesions under the influence of chemokines. Furthermore, it supports real-time high-resolution imaging, enabling quantitative analysis at the single-cell level of immune synapse formation and the reverse regulatory mechanisms of the organoid microenvironment. This significantly improves the clinical predictability of immunotherapy evaluation and drug screening.

[0063] The dimensions of the cell interception structure and microgroove structure described in this invention can be finely adjusted according to the size of the target cells, making it suitable for adherent cells with a diameter of 8-20 μm, such as immune cells, stem cells, epithelial cells, and tumor cells. Furthermore, the chip structure is compatible with various fluid actuation methods (injection pump, gravity flow, liquid level difference, etc.), making it suitable for routine laboratory operations. In addition, the chip is inexpensive, has good reproducibility, and possesses significant potential for platform-based commercialization.

[0064] Where there is no conflict, the above embodiments and features described herein can be combined with each other.

[0065] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A cell communication microfluidic chip with a cell capture structure, characterized in that, include: The main channel consists of two parallel main channels spaced apart. One end of each main channel is configured as the first liquid inlet, and the other end is configured as the first liquid outlet. A cell interception structure is provided in each main channel. The cell interception structure has a groove on the flow-facing side of the main channel that can accommodate at least one cell to be cultured. The groove has at least one opening for culture medium to flow out only. as well as The culture medium exchange structure is located between the two main channels to allow for the exchange of culture medium between the two main channels.

2. The cell communication microfluidic chip with a cell capture structure according to claim 1, characterized in that, The cell interception structures are arranged in rows along the flow direction of the culture medium in the main channel, with at least one cell interception structure in each row. The cell interception structures in adjacent rows are staggered, and the gap between two cell interception structures in the upstream row is directly opposite the cell interception structure in the adjacent downstream row.

3. The cell communication microfluidic chip with a cell capture structure according to claim 1, characterized in that, The cell interception structure is formed by two L-shaped blocking structures with a gap between them. A groove is formed between the two arms of the two L-shaped blocking structures along the flow direction, and an opening is formed between the two arms of the two L-shaped blocking structures perpendicular to the flow direction.

4. The cell communication microfluidic chip with a cell capture structure according to claim 1, characterized in that, The culture medium interaction structure is a grid-like microchannel that directly connects two main channels. The grid-like microchannel is composed of several microgrooves arranged at intervals along the fluid flow direction. The size of the microgrooves is configured to allow only the culture medium to pass through.

5. The cell communication microfluidic chip with a cell capture structure according to claim 4, characterized in that, The width of the microgroove is 2-10 micrometers.

6. The cell communication microfluidic chip with a cell capture structure according to claim 1, characterized in that, The culture medium interaction structure includes a grid-like microchannel connected to the opposite side of each main channel and a cell culture communication layer connecting two grid-like microchannels.

7. The cell communication microfluidic chip with a cell capture structure according to claim 6, characterized in that, The cell culture communication layer includes a group of culture chambers connected to the main channel via a grid-like microchannel and a central exchange chamber connected to the two groups of culture chambers via branch channels.

8. The cell communication microfluidic chip with a cell capture structure according to claim 7, characterized in that, The culture chambers in the culture chamber group are connected in series, and each culture chamber is connected to the corresponding main channel through a grid-like microchannel.

9. The cell communication microfluidic chip with a cell capture structure according to claim 8, characterized in that, The first and last culture chambers in the culture chamber group are respectively equipped with a second liquid inlet and a second liquid outlet.

10. The application of the cell communication microfluidic chip according to any one of claims 1-9 in non-contact cell co-culture, characterized in that, Used for research on cell communication mediated by secretory factors between different cell lines.

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