Microfluidic-mediated static and dynamic synergistic cell culture chip and application thereof

Abstract

The invention relates to a micro-fluidic mediated static and dynamic collaborative cell culture chip and application thereof, relates to the technical field of biotechnology, and aims to solve one of the problems that in the prior art, a real mechanical microenvironment in a body is difficult to re-engrave through static culture, and a chip device capable of synchronously implementing static and mechanical stretching dynamic biphase culture is lacked. The invention discloses a microfluidic-mediated static and dynamic collaborative cell culture chip and application thereof. The microfluidic-mediated static and dynamic collaborative cell culture chip comprises a cell culture chip and a pneumatic system matched with the cell culture chip for use, the cell culture chip comprises a micro-flow regulation and control chamber and a culture chamber, and the pneumatic system adjusts air pressure in the micro-flow regulation and control chamber to drive local movement of the bottom surface of the culture chamber, so that to-be-cultured cells are simultaneously subjected to dynamic culture and static culture in the culture chamber. Static culture and dynamic culture can be synchronously carried out in the same chip, an in-vivo mechanical microenvironment is effectively copied, and static and mechanical stretching dynamic biphase culture is synchronously implemented.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a microfluidic-mediated static and dynamic synergistic cell culture chip and its applications. Background Technology

[0002] The traditional technical approach in cell and tissue culture can be summarized in three core characteristics: first, the carrier medium mainly consists of culture dishes and multi-well plates; second, the culture mode promotes static two-dimensional culture; and third, the regulation method relies on active components such as growth factors in the culture medium to regulate cell physiological activities. The rise of microfluidic chip technology has revolutionized this traditional approach—the cross-disciplinary integration of the two has become a core development trend in the field. "Lab-on-a-chip" is a typical term for microfluidic chips, whose core advantages lie in: the ability to precisely manipulate fluids within a micrometer-level space, condensing and integrating traditional laboratory functions such as chemical analysis and biological detection onto a chip of only a few square centimeters; and possessing a series of characteristics including low material consumption, minimal reagent usage, high reaction efficiency, excellent detection sensitivity, high portability, ease of operation, and flexible combination of unit technologies.

[0003] Polydimethylsiloxane (PDMS) is the mainstream material for fabricating such chips. Its excellent light transmittance, gas permeability, biocompatibility, and flexibility give it unique advantages, enabling its widespread application in research fields such as biochemistry and molecular biology. Using these PDMS chips, researchers can not only construct composite culture microenvironments that closely resemble the in vivo growth state of cells and tissues, but also precisely control key mechanical parameters within the microenvironment; simultaneously, the dynamic response of cells and tissues to changes in microenvironment parameters can be tracked and observed in real time.

[0004] Currently, there are still many shortcomings in the relevant technological system, which are specifically reflected in the following two aspects:

[0005] 1. The crucial regulatory role of biomechanical factors in cell and tissue behavior and function has not been considered in traditional static two-dimensional culture systems. Therefore, the true in vivo mechanical microenvironment cannot be replicated, ultimately leading to a significant deviation between in vitro culture results and in vivo physiological states, making it difficult to accurately reflect the natural physiological characteristics of cells.

[0006] 2. Neither purely dynamic nor static culture modes can guarantee the stability and uniformity of microenvironment parameters—fluctuations easily occur in both the concentration of nutrients such as growth factors in the culture medium and mechanical parameters such as fluid shear force. The direct impact of these fluctuations is that researchers cannot efficiently and accurately analyze the differences in how mechanical stimulation and static culture regulate cellular physiological functions. This problem not only restricts in-depth research into related regulatory mechanisms but also reduces the efficiency and reliability of drug screening results.

[0007] In summary, the most pressing technical challenge for researchers in this field is to develop a tissue chip that can simultaneously achieve static culture and dynamic culture under mechanical traction on the same chip platform. Summary of the Invention

[0008] Based on the above analysis, the present invention aims to provide a microfluidic-mediated static and dynamic synergistic cell culture chip and its application, in order to solve one of the problems in the prior art, such as the difficulty in replicating the real mechanical microenvironment in vivo by static culture, the lack of chip devices that can simultaneously carry out static and mechanical stretch dynamic biphasic culture, and the difficulty in accurately defining the differential effects of stretch mechanical stimulation and static culture on cell physiological regulation due to uneven microenvironment parameters.

[0009] The objective of this invention is mainly achieved through the following technical solutions: A microfluidic-mediated static and dynamic synergistic cell culture chip includes a cell culture chip and a pneumatic system used in conjunction with the cell culture chip; the cell culture chip includes a culture layer, a transmission layer and a microfluidic bottom layer; the culture layer has a culture chamber, the culture chamber includes a static culture area and a dynamic culture area, and the microfluidic bottom layer has a microfluidic control chamber connected to the pneumatic system; The bottom surface of the culture layer, the transmission layer, and the top surface of the microfluidic bottom layer are bonded together by plasma bonding. The pneumatic system adjusts the air pressure in the microfluidic control chamber and drives the local movement of the bottom surface of the culture chamber through the transmission layer, so that the cells to be cultured can be dynamically and statically cultured simultaneously in the culture chamber.

[0010] Furthermore, the culture chamber is arranged opposite to the microfluidic control chamber.

[0011] Furthermore, the width of the culture chamber is greater than the width of the microfluidic control chamber, and the length of the culture chamber is less than the length of the microfluidic control chamber.

[0012] Furthermore, the transmission layer is made of a super-elastic PDMS film material.

[0013] Furthermore, the bottom surface of the culture chamber and the top surface of the microfluidic sublayer are both made of PDMS material.

[0014] In another aspect, the present invention provides a microfluidic-mediated method for the co-culture of cells in static and dynamic states, which utilizes the aforementioned cell culture chip to perform static and dynamic co-culture of cells.

[0015] Furthermore, the method includes the following steps: Step 1: Place the cells to be cultured onto the culture chamber; Step 2: Use a pneumatic system to change the air pressure value in the microfluidic control chamber, so as to increase or decrease the air pressure in the microfluidic control chamber. Step 3: The top layer of the microfluidic control chamber drives the dynamic culture zone of the culture chamber to move upward or downward through the transmission layer, maintaining the traction stimulation culture of the cells laid in the dynamic culture zone; at the same time, the cells laid in the static culture zone are statically cultured.

[0016] Furthermore, in step two, the pneumatic system reduces the air pressure in the microfluidic control chamber by 2 kPa relative to atmospheric pressure.

[0017] Furthermore, in step two, the pneumatic system increases the air pressure in the microfluidic control chamber by 2 kPa relative to atmospheric pressure.

[0018] Furthermore, between step one and step two, the pressure change value of the pneumatic system regulating microfluidic control chamber is determined based on the preset traction amplitude of the transmission layer.

[0019] Compared with the prior art, the present invention can achieve at least one of the following beneficial effects: (1) The microfluidic-mediated static and dynamic synergistic cell culture chip of the present invention includes a cell culture chip and a pneumatic system, which are used in conjunction with the cell culture chip. The cell culture chip includes a culture layer, a transmission layer and a microfluidic bottom layer. The culture layer has a culture chamber, which includes a static culture area and a dynamic culture area. The pneumatic system adjusts the air pressure in the microfluidic control chamber and drives the local movement of the bottom surface of the culture chamber through the transmission layer, so that the cells to be cultured can be cultured dynamically and statically in the culture chamber at the same time, realizing the synchronous development of static and dynamic culture in the same chip. The layered bonding structure ensures the sealing and stability of the culture environment. The pneumatic system accurately transmits mechanical stimulation, effectively replicating the in vivo mechanical microenvironment. It provides a reliable experimental carrier to clarify the differences in cell physiological regulation between the two culture modes in the same environment, and greatly improves the efficiency of related mechanism research.

[0020] (2) The microfluidic-mediated static and dynamic synergistic cell culture chip of the present invention has a culture chamber and a microfluidic control chamber arranged opposite to each other. This ensures that the air pressure changes generated by the pneumatic system directly and efficiently act on the transmission layer, reducing mechanical transmission loss and improving the accuracy and response speed of the traction stimulation in the dynamic culture area. By optimizing the width-to-length ratio of the two chambers, the corresponding areas of the culture chamber and the microfluidic control chamber can move upward or downward due to the air pressure changes in the microfluidic control chamber and the traction effect of the top layer of the microfluidic control chamber, forming a dynamic culture area. At the same time, other areas of the culture chamber will not be affected by the air pressure changes in the microfluidic control chamber, that is, the remaining areas form a static culture area for static cell culture, so that the dynamic culture area obtains sufficient traction stroke, reducing the possibility of the microfluidic control chamber over-occupying chip space, and making the air pressure force distribution more uniform. At the same time, there is a static culture area in the same culture chamber that is not affected by traction, solving the problem that in the prior art, the tissue chip cannot realize static culture and mechanical traction dynamic culture simultaneously on the same chip platform. Attached Figure Description

[0021] The accompanying drawings are for illustrative purposes only and are not intended to limit the invention. Throughout the drawings, the same reference numerals denote the same parts.

[0022] Figure 1 This is a schematic diagram of the structure of the microfluidic-mediated static and dynamic synergistic cell culture chip of Embodiment 1 of the present invention; Figure 2 This is one of the structural schematic diagrams of the cell culture chip in Embodiment 1 of the present invention; Figure 3 This is a second schematic diagram of the cell culture chip structure in Embodiment 1 of the present invention; Figure 4 This is a schematic diagram of the structure of the PDMS thin film material in Embodiment 2 of the present invention; Figure 5 The multi-dimensional mechanical property test data and the fitting curve of the new Hooke constitutive model of the PDMS thin film material described in Example 2 of the present invention are shown. Figure 6 This is a load-constraint diagram illustrating the static structural analysis of the PDMS thin film material described in Embodiment 2 of the present invention. Figure 7 This is a finite element mesh generation model diagram of the PDMS thin film material described in Embodiment 2 of the present invention; Figure 8 This is one of the total deformation cloud diagrams for the static structural analysis of the PDMS thin film material described in Embodiment 2 of the present invention; Figure 9This is the second total deformation cloud diagram of the static structural analysis of the PDMS thin film material described in Embodiment 2 of the present invention; Figure 10 This is one of the X-axis oriented deformation cloud diagrams for the static structural analysis of the PDMS thin film material described in Embodiment 2 of the present invention; Figure 11 This is the second X-axis oriented deformation cloud map of the PDMS thin film material described in Embodiment 2 of the present invention for static structural analysis; Figure 12 Equivalent stress (Feng) for static structural analysis of the PDMS thin film material described in Example 2 of the present invention One of the Mises stress contour maps; Figure 13 Equivalent stress (Feng) for static structural analysis of the PDMS thin film material described in Example 2 of the present invention Mises stress contour map II; Figure 14 The third total deformation cloud diagram for the static structural analysis of the PDMS thin film material described in Embodiment 2 of the present invention. Figure 15 The X-axis oriented deformation contour map of the PDMS thin film material described in Embodiment 2 of the present invention is shown. Figure 16 This is a flowchart of Embodiment 3 of the present invention; Figure 17 This is a graph showing the relationship between PDMS film deformation and vacuum degree in Example 3 of the present invention; Figure 18 This is a cell viability diagram of the static culture area in Example 3 of the present invention; Figure 19 This is a statistical diagram of the cell arrangement direction in the static culture area in Example 3 of the present invention; Figure 20 This is a cell activity diagram of the dynamic culture area in Example 3 of the present invention; Figure 21 This is a statistical diagram of the cell arrangement direction in the dynamic culture area in Example 3 of the present invention; Figure 22 This is a cell activity diagram of the static channel region in Example 3 of the present invention; Figure 23 This is a statistical diagram of the cell arrangement direction in the static channel region in Embodiment 3 of the present invention; Figure 24 This is a flowchart of the experiment on microfluidic-mediated static and dynamic synergistic cell culture chip and mechanical stress stimulation in Embodiment 4 of the present invention.

[0023] Figure label: 1-Cell culture chip, 11-Culture layer, 111-Culture chamber, 1111-Static culture area, 1112-Dynamic culture area, 12-Transmission layer, 13-Microfluidic bottom layer, 131-Microfluidic control chamber; 2-Pneumatic system. Detailed Implementation

[0024] Preferred embodiments of the present invention will now be described in detail with reference to the accompanying drawings, which form part of this application and are used together with the embodiments of the present invention to illustrate the principles of the present invention, but are not intended to limit the scope of the present invention.

[0025] Example 1 For the research scenario of cell physiological regulation mechanism, this embodiment is mainly used to solve one of the following technical problems in the existing technical solutions: the static culture mode is difficult to reproduce the physiological mechanical microenvironment in vivo, and there is a lack of dedicated chip carriers that can simultaneously carry out static incubation and mechanical stretching dynamic culture; at the same time, the lack of uniformity of the culture system microenvironment makes it impossible to accurately distinguish the differences in the regulation of cell physiological activities by mechanical stretching mechanical stimulation and static culture.

[0026] One specific embodiment of the present invention discloses a microfluidic-mediated static and dynamic synergistic cell culture chip, such as... Figure 1 As shown, it includes a cell culture chip 1 and a pneumatic system 2 used in conjunction with the cell culture chip 1; the cell culture chip 1 includes a culture layer 11, a transmission layer 12, and a microfluidic bottom layer 13; as Figure 2 and Figure 3 As shown, the culture layer 11 has a culture chamber 111, which includes a static culture area 1111 and a dynamic culture area 1112. The microfluidic bottom layer 13 has a microfluidic control chamber 131 connected to the pneumatic system 2.

[0027] The bottom surface of the culture layer 11, the transmission layer 12, and the top surface of the microfluidic bottom layer 13 are bonded together by plasma bonding. The pneumatic system 2 adjusts the air pressure in the microfluidic control chamber 131 and drives the local movement of the bottom surface of the culture chamber 111 through the transmission layer 12, so that the cells to be cultured can be dynamically and statically cultured in the culture chamber at the same time.

[0028] This invention relates to a microfluidic-mediated static and dynamic synergistic cell culture chip, comprising a cell culture chip 1 and a pneumatic system 2, which works in conjunction with the cell culture chip 1. The cell culture chip 1 includes a culture layer 11, a transmission layer 12, and a microfluidic bottom layer 13. The culture layer 11 has a culture chamber 111, which includes a static culture area 1111 and a dynamic culture area 1112. The pneumatic system 2 adjusts the air pressure within the microfluidic control chamber 131, and through the transmission layer 12, drives local movement of the bottom surface of the culture chamber 111, allowing the cells to be cultured to undergo simultaneous dynamic and static culture within the culture chamber. This enables simultaneous static and dynamic culture within the same chip, ensures the airtightness and stability of the culture environment through a layered bonding structure, and precisely transmits mechanical stimuli using the pneumatic system 2, effectively replicating the in vivo mechanical microenvironment. It provides a reliable experimental platform for clarifying the differences in cell physiological regulation between the two culture modes within the same environment, significantly improving the efficiency of related mechanism research.

[0029] Furthermore, the pneumatic system 2 includes a cylinder and a pressure control component. The output end of the cylinder is connected to the microfluidic control chamber 131, and the cylinder's air path interface is connected to the solenoid valve outlet of the pressure control system via a high-pressure air pipe. The PLC in the pressure control component controls the switching of the solenoid valve, thereby lowering and raising the air pressure within the microfluidic control chamber 131. When the air pressure within the microfluidic control chamber 131 decreases, the top layer of the microfluidic bottom layer 13 causes the bottom surfaces of the transmission layer 12 and the culture layer 11 to deform downwards under atmospheric pressure. When the air pressure within the vacuum chamber increases and returns to atmospheric pressure, the top layer of the microfluidic bottom layer 13 causes the bottom surfaces of the transmission layer 12 and the culture layer 11 to return to their original deformation state.

[0030] The bottom surface of the culture layer 11, the transmission layer 12, and the top surface of the microfluidic bottom layer 13 are bonded together by plasma bonding, which not only ensures the sealing performance of the chip but also reduces the probability of leakage of culture medium in the culture chamber 111 and air leakage in the microfluidic control chamber 131.

[0031] Furthermore, such as Figure 2 and Figure 3As shown, the culture chamber 111 and the microfluidic control chamber 131 are arranged opposite each other. The width of the culture chamber 111 is greater than the width of the microfluidic control chamber 131, and the length of the culture chamber 111 is less than the length of the microfluidic control chamber 131. The microfluidic control chamber 131 is located at the center directly below the culture chamber 111, and the length of the microfluidic control chamber 131 is longer than the length of the culture chamber 111. That is, the microfluidic control chamber 131 can form a dynamic culture zone 1112 in the central region of the culture chamber 111 that moves with the change of air pressure inside the microfluidic control chamber 131, and form a static culture zone 1111 in other areas of the culture chamber 111 that is not affected by the air pressure inside the microfluidic control chamber 131, so that the pneumatic system 2 can precisely drive the deformation of the dynamic culture zone 1112. This technology enables the simultaneous construction of dynamic culture zone 1112 and static culture zone 1111 within the same cell culture chamber 111, ensuring complete consistency in the microenvironment, including nutrient concentration and fluid shear force, except for mechanical traction. It solves the experimental error problem caused by the separation of biphasic culture in traditional techniques and significantly improves the reliability and intuitiveness of research related to traction mechanical stimulation.

[0032] Furthermore, the microfluidic control chamber 131 is a cuboid groove, for example, the size of the microfluidic control chamber 131 is 53 mm. 6mm A rectangular groove with a 4mm rounded corner (R=1mm); correspondingly, the culture chamber 111 is also a rectangular groove, for example, the size of the culture chamber 111 is 36mm. 18mm A rectangular groove with 4mm rounded corners (R=1mm).

[0033] Furthermore, the culture chamber 111 also includes a cell culture medium inlet and a cell culture medium outlet. The cell culture medium inlet and outlet provide a convenient nutrient injection channel for the culture chamber 111, allowing for the rapid and uniform delivery of culture medium, growth factors, and other nutrients to the static and dynamic culture zones 1112. This design ensures that the two-phase culture zones receive sufficient nutrients simultaneously, avoiding differences in cell growth caused by uneven nutrient supply, further guaranteeing the consistency of the microenvironment except for traction stimulation, and improving the accuracy of experimental comparisons.

[0034] Furthermore, the cell culture medium inlet and outlet are also rectangular grooves, with the cell culture medium inlet measuring 4 mm. 2mm 4mm, the size of the cell culture medium outlet is 4mm. 2mm 4mm.

[0035] Furthermore, the transmission layer 12 is a superelastic PDMS membrane. Superelastic PDMS membranes possess superior deformation capacity and resilience compared to ordinary PDMS membranes, enabling them to undergo uniform and controllable tensile deformation under pressure changes, and they are less prone to fatigue damage after repeated stretching. This characteristic ensures the stability and repeatability of mechanical stretching stimulation in the dynamic culture zone 1112, reduces the probability of uneven stretching force or structural damage due to insufficient membrane elasticity, and ensures the long-term stable conduct of comparative experiments between dynamic and static cultures.

[0036] Furthermore, the thickness of the transmission layer 12 is 100 μm, and the Young's modulus of the transmission layer 12 is 1.71 MPa. For example... Figure 3 As shown, the thickness of the transmission layer 12 is 2100 μm, and its Young's modulus is 1.71 MPa. This design ensures that the transmission layer 12 is sensitive to changes in air pressure (effective deformation can be generated with slight adjustments in air pressure) while also possessing sufficient structural strength to withstand repeated traction cycles. This avoids the problems of the transmission layer 12 being too thin and prone to tearing, or too thick and insufficient deformation. This parameter design allows for precise control of the mechanical traction force, perfectly adapting to the mechanical stimulation requirements of different cell types, and improving the versatility of the chip and the accuracy of experimental data.

[0037] Furthermore, the bottom surface of the culture layer 11 and the top surface of the microfluidic bottom layer 13 are both made of PDMS material. The bottom surface of the culture layer 11, the transmission layer 12 and the top surface of the microfluidic bottom layer 13 are all made of PDMS material, and the bottom surface of the culture layer 11, the transmission layer 12 and the top surface of the microfluidic bottom layer 13 are bonded together by plasma bonding.

[0038] Furthermore, the cell culture chip 1 also includes a chip clamp; the chip clamp is used to fix the culture layer 11, the transmission layer 12 and the microfluidic bottom layer 13. The chip clamp is set to ensure that the culture layer 11, the transmission layer 12 and the microfluidic bottom layer 13 are firmly fixed, preventing relative displacement of the three-layer structure during air pressure changes and stretching, and ensuring bonding sealing.

[0039] Furthermore, the chip fixture connects the output of the pneumatic system 2 to the microfluidic control chamber 131, and fixes the output of the pneumatic system 2 to the microfluidic control chamber 131. The chip fixture ensures stable communication between the microfluidic control chamber 131 and the pneumatic system 2, preventing leakage or pressure loss during air pressure transmission. This design provides dual protection for the overall structural stability of the chip and the precision of pneumatic control, ensuring the reliable implementation of the dynamic traction function.

[0040] Furthermore, the chip fixture also includes fixing screws that pass through the culture layer 11, the transmission layer 12, and the microfluidic bottom layer 13 to fix the culture layer 11, the transmission layer 12, and the microfluidic bottom layer 13 together. The chip fixture is a cuboid made of acrylic material, and its through holes are made using a laser engraving machine; this chip fixture is used to fix the multi-force field coupled cell culture chip 1 to the pneumatic system 2.

[0041] Example 2 To examine the mechanical properties of PDMS (chloroprene rubber film) and its material information, and to verify whether PDMS is suitable for use in static and dynamic synergistic cell culture chip 1, this embodiment, based on Embodiment 1, further verifies the mechanical suitability of PDMS as an elastic component (transmission layer 12).

[0042] 1. Visualize key simulation parameters of thin films The PDMS film material is displayed using segmented labeling (6mm x 3 segments), such as... Figure 4 As shown, the parameters of this PDMS thin film material include: Model attributes: 3D model, Beam type, Flexible stiffness (flexibility); Geometric parameters: Thickness 0.2mm (Manual mode, offset type Middle); Material properties: Assigned as neoprene rubber; Physical effects: enabling nonlinear effects to adapt to the properties of hyperelastic materials and thermal strain effects.

[0043] By simulating the basic parameters of the thin film, it is determined that the properties of the ANSYS simulation model are consistent with those of the actual processed thin film (such as thickness, material, and flexibility), providing parameter support for the accuracy of subsequent mechanical simulations.

[0044] 2. Multidimensional mechanical testing of PDMS thin film materials. The mechanical properties of PDMS thin film materials are presented in the form of stress-strain curves, such as... Figure 5 As shown: Biaxial Test Data: Stress changes at strains of 0 to 1.0; Shear test (ShearTestData): Shear stress distribution at strains of 0~4.4e-1; Uniaxial Test Data: Tensile stress at strain 0~1.6e+0; Volumetric Test Data: The relationship between volumetric strain and stress; Material model annotation: The Neo-Hookean model is used (a constitutive model commonly used for hyperelastic materials, adapted to the nonlinear elastic properties of chloroprene rubber).

[0045] 3. Perform load-constraint analysis on PDMS thin film material.

[0046] The boundary conditions and loads simulated by ANSYS are presented in a two-dimensional schematic diagram, such as... Figure 6 and Figure 7 As shown: Constraints: Point A on the left is FixedSupport (a fixed constraint that simulates the actual fixing method at both ends of the thin film); Load: Apply Pressure = 2e-003MPa (i.e., 2kPa, corresponding to the pressure change in the vacuum chamber of the microfluidic chip) at point B on the right. Coordinate system: X / Z axis markings, clearly indicating the load direction and geometric orientation (the air pressure load acts along the Z-axis in the middle of the film).

[0047] Load-constraint analysis was used to reconstruct the actual stress scenario of the transmission layer 12 in the microfluidic chip (the load of the thin film on the gas pressure change in the chip vacuum chamber, and the fixed constraints between the two ends of the thin film and the upper and lower layers of the chip); the working condition boundary of the simulation was clarified to ensure that the simulation working condition is consistent with the actual application scenario (such as the tensile stimulation parameter of the chip corresponding to 2 kPa gas pressure), which provides a prerequisite for the effectiveness of subsequent deformation and stress analysis.

[0048] 4. Visual analysis of deformation distribution in PDMS thin film materials In the total deformation contour plot of the thin film, as shown Figure 8 and Figure 9 As shown, the three-dimensional total deformation distribution of the film under a 2 kPa load is presented using a color gradient (from blue to red): the deformation range is 0~2.8232 mm (red is the maximum value, located in the load application area in the middle of the film; blue is the minimum value, located in the fixed constraint area); the maximum deformation of the film (2.8232 mm) is quantified to verify that it can meet the amplitude requirements of cell traction in the dynamic culture area 1112 of the microfluidic chip, and the deformation distribution pattern is intuitively displayed (large in the middle and small at both ends), indicating that there is no local deformation anomaly, which shows that the load is uniformly transferred.

[0049] In the X-axis oriented deformation contour map of the thin film, as shown... Figure 10 , Figure 11 , Figure 14 and Figure 15 As shown, the directional deformation distribution of the thin film along the X-axis is presented using a color gradient, with a maximum value of 2.7114 mm (center) and a minimum value of 0 (fixed end). The coordinate system is related to the total deformation. Figure 1To achieve this, we focus on deformation data along the stretching direction (the main direction of cell stretching in the chip), eliminating interference from deformations in other directions, and more accurately matching the unidirectional stimulation requirements of cell stretching; we also verify the uniformity of deformation in the X direction to ensure that the stretching amplitude experienced by cells in the dynamic culture area is consistent.

[0050] 5. Stress Distribution Analysis of PDMS Thin Film Materials In the equivalent stress contour map of a thin film, the distribution of equivalent stress on the upper and lower surfaces of the film is presented using color gradients, such as... Figure 12 and Figure 13 As shown, the stress range is 0~0.086347MPa (red indicates the maximum value, located in the transition zone between the middle of the film and the fixed end; blue indicates the minimum value, located at the fixed end). The maximum stress of the film (0.086347MPa) is quantified. This stress is much lower than the breaking strength of neoprene rubber, ensuring that the film will not break under a 2kPa load, thus meeting the structural safety requirements for long-term cyclic tension.

[0051] Example 3 Furthermore, in order to solve the technical problem of how to simultaneously perform dynamic and static culture of cells to be cultured in the same culture chamber 111, and to improve the operability and experimental reproducibility of cell culture using chips.

[0052] This embodiment further provides a microfluidic-mediated method for the co-culture of cells in static and dynamic states, based on embodiment 1. This method uses the cell culture chip 1 described above to perform static and dynamic co-culture of cells.

[0053] The culture layer 11, transmission layer 12, and microfluidic bottom layer 13 have an irreversible bonded structure, and the microfluidic control chamber 131 has the same microfluidic pathway, realizing the integrated synergy of static and dynamic culture: the same culture medium nutrient supply, fluid shear force, and temperature environment are provided in the same chip. The static culture zone 1111 and the dynamic culture zone 1112 use mechanical traction stimulation as the only variable, which completely solves the experimental error caused by inconsistent microenvironment parameters in traditional separate device culture, and improves the credibility of the conclusions on the difference in cell physiological regulation by traction mechanics. The PDMS material used in the cell culture chip 1 (including the superelastic PDMS transmission layer 12) has excellent biocompatibility (non-cytotoxic) and light transmittance, which not only ensures the cell adhesion rate, but also supports real-time microscopic observation of the dynamic response of cells in biphasic culture, and data can be obtained without interrupting the culture.

[0054] Furthermore, the microfluidic-mediated co-culture method for static and dynamic cell culture includes the following steps: Figure 16 As shown: Step 1: Place the cells to be cultured onto culture chamber 111; Step 2: Use the pneumatic system 2 to change the air pressure value in the microfluidic control chamber 131, so that the air pressure in the microfluidic control chamber 131 increases or decreases. Step 3: The top layer of the microfluidic control chamber 131 drives the dynamic culture zone 1112 of the culture chamber 111 to move upward or downward through the transmission layer 12, thereby maintaining the traction stimulation culture of the cells laid in the dynamic culture zone 1112; at the same time, the cells laid in the static culture zone 1111 are statically cultured.

[0055] The culture method described in this embodiment involves simultaneous initiation and execution of biphasic culture, allowing for the acquisition of cell morphology, proliferation, and differentiation data under two culture modes at the same time and in the same culture environment. This avoids the time difference errors caused by traditional staged culture, shortens the research cycle, and captures the immediate response of cells to traction stimulation. The cell culture steps in this embodiment are simple and controllable, requiring no complex manual intervention. Ordinary laboratory personnel can quickly learn to use it, solving the problem of cumbersome traditional traction culture operations.

[0056] During cell culture, the pressure changes in the microfluidic control chamber 131 are precisely converted into mechanical tension through the transmission layer 12 (hyperelastic PDMS membrane). Furthermore, the static culture area 1111 is irreversibly bonded to the PDMS in the microfluidic control chamber 131, ensuring that the cells in the static culture area 1111 are free from additional mechanical interference. This achieves strict control of a single variable and makes the attribution of differences in cell growth more direct.

[0057] Furthermore, in step two, the pneumatic system 2 reduces the air pressure in the microfluidic control chamber 131 by up to 2 kPa relative to atmospheric pressure.

[0058] The air pressure parameters of the pneumatic system 2 are matched with the mechanical properties of the hyperelastic PDMS membrane and the ANSYS simulation data. When the air pressure is reduced by 2 kPa, the maximum total deformation of the transmission layer 12 (hyperelastic PDMS membrane) reaches 2.8232 mm, the X-direction directional deformation reaches 2.7114 mm, and the maximum equivalent stress is 0.086347 MPa. This tensile strength is within the physiological tolerance range of cells, will not cause cell damage, and can effectively trigger the cell mechanical response.

[0059] The pressure change of 2 kPa is stable and easy to control. Combined with the high fitting degree between the medium variable and the vacuum degree in the chip characterization data (R²=0.9941), the consistency of tensile strength in different batches of experiments is ensured, and the repeatability of the experiment is improved. Downward deformation mode can simulate the positive mechanical load borne by cells in vivo, making the functional phenotype of in vitro cultured cells closer to the real state in vivo, and providing a reliable in vitro model for related mechanism research.

[0060] Furthermore, in step two, the pneumatic system 2 increases the air pressure in the microfluidic control chamber 131 by 2 kPa relative to atmospheric pressure.

[0061] When the air pressure increases by 2 kPa, the transmission layer 12 undergoes an upward controllable deformation. The deformation amount is symmetrically distributed with the downward deformation, forming a reverse mechanical stimulus and enriching the diversity of the traction mode. The equivalent stress of the PDMS membrane corresponding to this air pressure value is ≤0.086347 MPa. The deformation process is mild and reversible. After the pressurization is stopped, the PDMS membrane can quickly recover its flatness, supporting long-term cyclic traction culture without causing continuous mechanical damage to the cells. When combined with the air pressure reduction mode, bidirectional alternating traction can be achieved, simulating the periodic bidirectional force environment of cells in vivo.

[0062] Furthermore, between step one and step two, the pneumatic system 2 adjusts the air pressure change value of the microfluidic control chamber 131 based on the preset traction amplitude of the transmission layer 12.

[0063] Establish a precise correspondence between stretching amplitude and air pressure value to solve the problem of the inability to quantify stimulation intensity in traditional stretching culture.

[0064] The PDMS membrane stretching amplitude can be preset according to experimental needs, and the required air pressure change value can be derived in reverse to realize personalized stretching scheme design and adapt to the different needs of different cell types for mechanical stimulation. The preset parameters can be stored and reused, making the experimental data of different laboratories more comparable when operating according to the preset parameters, providing support for the establishment of standardized experimental procedures, while avoiding insufficient stretching or cell damage caused by blindly adjusting the air pressure.

[0065] like Figure 17 As shown, the fitting formula for the relationship between PDMS film type variable and vacuum degree is: Formula (1): y = 59.80x² + 10.16x + 1.851 Where y represents the stretching amplitude of the PDMS film (%, percentage deformation), and x represents the vacuum degree; Formula (2) for x = ΔP / P0 Formula (3) Wherein, ΔP represents the pressure change of the microfluidic control chamber 131 relative to the atmospheric pressure, P1 represents the atmospheric pressure inside the microfluidic control chamber 131, and P0 represents the standard atmospheric pressure.

[0066] When different cell cultures are performed, it is known that different mechanical stretching amplitudes, i.e. PDMS membrane stretching amplitudes, need to be provided to the cells to be cultured in the dynamic culture zone 1112 within the culture chamber. The atmospheric pressure in the corresponding microfluidic control chamber 131 is calculated using the above formulas (1), (2) and (3). During cell culture, the atmospheric pressure in the microfluidic control chamber 131 can be accurately controlled by the pneumatic system 2 to achieve the PDMS membrane stretching amplitude in the dynamic culture zone 1112, realize the predetermined mechanical stretching stimulation, improve the reliability of the stretching stress stimulation study, and provide a stable and reproducible mechanical microenvironment for the analysis of cell response mechanisms.

[0067] Example 4: Validation of the effects of static-dynamic co-culture and traction regulation of cells based on microfluidic chips This study aims to verify the regulatory effect of microfluidic-mediated static and dynamic synergistic cell culture chips on cell activity, alignment, and morphological phenotype under single-variable control, and to clarify the reliability and practicality of the chip for cell mechanical response research.

[0068] 1. Experimental Materials and Equipment Cell sample: Primary mouse ligamentum flavum cells (MLFCs) Main experimental equipment, consumables, and reagents:

[0069] 2. Experimental Procedure Cell seeding and culture in planar dynamic cell culture chips, such as Figure 24 As shown: (1) After UV sterilization, the planar dynamic cell culture chip was transferred to a clean bench. Using a sterile syringe, 1 mL of 0.01% rat tail collagen I solution was slowly infused into the chip culture chamber. After tightening the cap of the Luer connector, the chip was transferred to a forced-air oven and incubated at 60°C for 6 h. (2) Remove the incubated planar dynamic cell culture chip and use a syringe to aspirate the residual rat tail collagen solution inside the chamber. Use a sterile syringe to draw approximately 1 mL of PBS buffer and pour it into the chip culture chamber. Rinse slowly 3 times before use. (3) Adjust the cell suspension density to 1×10⁶ / mL, and slowly perfuse the cell suspension into the planar dynamic cell culture chip using a syringe. Tighten the cap of the Luer connector. Transfer the chip to a CO₂ cell culture incubator and incubate statically for 24 hours. After the chip is dried, it is carefully removed and transferred to a UV sterilizer for sterilization and later use. The chip was secured to the slide of the thin-finger cylinder using a chip clamp and tightened with a screwdriver. The assembled system was then transferred to a CO2 cell culture incubator, and the dynamic strain cell culture system was run. The stretching parameters were set to an amplitude of 0–20% and a stretching frequency of 0.5 Hz. Uniaxial bidirectional stress stimulation was applied to the cells cultured in the chip, and the stretching culture was continued for 24 hours.

[0070] Notice: ① All the above injection operations should be performed as slowly as possible to avoid introducing too many air bubbles into the culture chamber; ② During all the above solution infusion operations, it is necessary to keep the chip as perpendicular to the table as possible so that air bubbles inside the chip can be better discharged from the other end.

[0071] 3. Sample testing and analysis: Cell viability assay: such as Figure 18 The static culture area 1111 was collected under a fluorescence microscope as shown. Figure 20 The image shows the fluorescence of cells in the dynamic culture area 1112 and as shown below. Figure 22 The fluorescence images of cells in the static channel region were acquired under a fluorescence microscope, and the viability of cells was statistically analyzed.

[0072] Cell arrangement and morphology analysis: such as Figure 19 , Figure 21 and Figure 23 As shown, the cell alignment rate is statistically analyzed.

[0073] 4. Experimental Results and Analysis 4.1. Cell viability verification Under continuous mechanical traction, the viable cell rate in the dynamic culture zone 1112 was not significantly different from that in the static culture zone 1111 (P>0.05), proving that the mechanical traction regulation of the chip does not damage cell viability and that the culture environment has good biocompatibility.

[0074] 4.2. Regulation of cell alignment Cells in static culture zone 1111 showed no obvious directional arrangement.

[0075] Cells in the dynamic culture zone 1112 showed a clear trend of directional arrangement, with the long axis of most cells parallel to the direction of traction (X-axis).

[0076] Using a single mechanical traction stimulus as a variable, cells were significantly induced to align themselves in the direction of traction, demonstrating that the chip can accurately realize the comparative study of static control and dynamic regulation, providing a reliable model for the analysis of cell mechanical response mechanisms.

[0077] 4.3. Changes in cell morphology and phenotype Mechanical traction mainly affects the aspect ratio of cell morphology, promoting cell differentiation towards elongation, while having no significant effect on cell spreading area. This indicates that traction stimulation has specific regulation of cell morphology, and the chip can achieve precise regulation and quantitative analysis of cell morphology phenotype.

[0078] This embodiment utilizes the chip's static-dynamic co-culture function, using mechanical traction as a single variable, to verify the chip's core performance: First, traction stimulation does not affect cell viability, and the culture system exhibits good biocompatibility; second, it can precisely induce cells to align along the traction direction, increasing the alignment rate by more than four times; and third, it can specifically regulate cell morphology and phenotype, enabling quantitative analysis of parameters such as aspect ratio. This solves the problems of difficult variable control and poor data reproducibility in traditional culture methods, and can be widely applied to basic research and applied development in fields such as cell mechanics and tissue engineering.

[0079] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention.

Claims

1. A microfluidic-mediated static and dynamic synergistic cell culture chip, characterized in that, The device includes a cell culture chip (1) and a pneumatic system (2) used in conjunction with the cell culture chip (1); the cell culture chip (1) includes a culture layer (11), a transmission layer (12) and a microfluidic bottom layer (13); the culture layer (11) has a culture chamber (111), the culture chamber (111) includes a static culture area (1111) and a dynamic culture area, and the microfluidic bottom layer (13) has a microfluidic control chamber (131) connected to the pneumatic system (2); The bottom surface of the culture layer (11), the transmission layer (12) and the top surface of the microfluidic bottom layer (13) are bonded together by plasma bonding. The pneumatic system (2) adjusts the air pressure in the microfluidic control chamber (131) and drives the local movement of the bottom surface of the culture chamber (111) through the transmission layer (12), so that the cells to be cultured can be cultured dynamically and statically at the same time in the culture chamber.

2. The microfluidic-mediated static and dynamic synergistic cell culture chip according to claim 1, characterized in that, The culture chamber (111) is arranged opposite to the microfluidic control chamber (131).

3. The microfluidic-mediated static and dynamic synergistic cell culture chip according to claim 2, characterized in that, The width of the culture chamber (111) is greater than the width of the microfluidic control chamber (131), and the length of the culture chamber (111) is less than the length of the microfluidic control chamber (131).

4. The microfluidic-mediated static and dynamic synergistic cell culture chip according to claim 1, characterized in that, The transmission layer (12) is made of ultra-elastic PDMS film material.

5. The microfluidic-mediated static and dynamic synergistic cell culture chip according to claim 1, characterized in that, The bottom surface of the culture layer (11) and the top surface of the microfluidic bottom layer (13) are both made of PDMS material.

6. A microfluidic-mediated method for the co-culture of cells in static and dynamic states, characterized in that, Cells are cultured statically and dynamically using the cell culture chip (1) according to any one of claims 1-5.

7. The method according to claim 6, characterized in that, Includes the following steps: Step 1: Place the cells to be cultured on the culture chamber (111); Step 2: Use the pneumatic system (2) to change the air pressure value in the microfluidic control chamber (131) to increase or decrease the air pressure in the microfluidic control chamber (131); Step 3: The top layer of the microfluidic control chamber (131) drives the dynamic culture area (1112) of the culture chamber (111) to move upward or downward through the transmission layer (12) to maintain the traction stimulation culture of the cells laid in the dynamic culture area (1112); at the same time, the cells laid in the static culture area (1111) are statically cultured.

8. The method according to claim 7, characterized in that, In step two, the pneumatic system (2) reduces the air pressure in the microfluidic control chamber (131) by 2 kPa relative to atmospheric pressure.

9. The method according to claim 7, characterized in that, In step two, the pneumatic system (2) increases the air pressure in the microfluidic control chamber (131) by 2 kPa relative to atmospheric pressure.

10. The method according to claim 7, characterized in that, Between step one and step two, the pneumatic system (2) adjusts the air pressure change value of the microfluidic control chamber (131) according to the preset pulling amplitude of the transmission layer (12).

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