Micro-fluidic chip for cell pulsating shear force environment and detection method

Abstract

The invention discloses a micro-fluidic chip for a cell pulsating shear force environment and a detection method in the technical field of cell biomechanics experiments.The micro-fluidic chip comprises a chip body and a control instrument, the chip body is provided with a fluid control channel and a cell culture cabin, and the fluid control channel is communicated with the cell culture cabin; the chip body is fixedly connected with the control instrument, the control instrument comprises a fluorescence microscope, an MEMS pressure sensor array, a plurality of micro-flow pumps and a plurality of air pumps, the MEMS pressure sensor array comprises a plurality of MEMS pressure sensors, and the micro-flow pumps are all communicated with the chip body. The invention aims to realize pulsation and unsteady state simulation of shear force, and integrates microenvironment conditions such as chemical gradient and substrate stiffness gradient with shear force simulation on the same experimental platform, so that the real microenvironment of cells in vivo is more comprehensively simulated, and the response mechanism of the cells to these mechanical and chemical signals is more accurately researched.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of cell biomechanics experiment, and particularly relates to a microfluidic chip for a cell pulsatile shear force environment and a detection method. BACKGROUND

[0002] In the field of the intersection and integration of biomedical engineering and micro-nano technology, the research on the cell mechanical microenvironment is promoting breakthrough progress in the directions of tissue engineering, disease model construction and drug screening. Among them, simulating the pulsatile shear force environment that periodically changes in the vascular system has become a key technical requirement for analyzing the function of vascular endothelial cells, the pathogenesis of vascular diseases and vascular regeneration medicine. Because the traditional macroscopic experimental system cannot accurately reproduce the physiological fluid dynamics characteristics, researchers have turned their attention to microfluidic technology platforms to achieve precise response research of cells to mechanical signals through micro-scale fluid control.

[0003] As a miniaturized integrated system, the core advantage of the microfluidic chip is that it can build a millimeter-scale channel network to generate a shear stress field with controllable spatial gradient and adjustable time dynamics in the cell culture area through a precisely designed microstructure and fluid driving device. The existing microfluidic chip can achieve simple control of shear force changes through pressure, but it often relies on constant flow pumps, making it difficult to simulate the pulsatile and non-steady shear force commonly seen in physiological conditions. Moreover, shear force often cooperates with microenvironment factors such as chemical gradient and substrate stiffness, and the existing technology, as shown in the patent document with the publication number CN108977359B, is limited by the structural design of the microfluidic chip, which can only design and control a single parameter in the same plane chamber, making it difficult to achieve independent and precise control of multiple parameters.

[0004] Therefore, it is necessary to propose a microfluidic chip for a cell pulsatile shear force environment and a detection method that can simulate pulsatile and non-steady shear force and integrate microenvironment conditions such as chemical gradient and substrate stiffness gradient with shear force simulation experiments. SUMMARY

[0005] To solve the above problems, the purpose of the present application is to provide a microfluidic chip for a cell pulsatile shear force environment and a detection method, which aims to simulate pulsatile and non-steady shear force and integrate microenvironment conditions such as chemical gradient and substrate stiffness gradient with shear force simulation on the same experimental platform, so as to more comprehensively simulate the real microenvironment of cells in vivo and more accurately study the response mechanism of cells to these mechanical and chemical signals.

[0006] In order to achieve the above object, the technical scheme of the present application is as follows: A microfluidic chip for cell pulsatile shear force environment, comprising a chip body and a control instrument, the chip body is provided with a fluid control channel and a cell culture cabin, the fluid control channel is communicated with the cell culture cabin, the chip body is fixedly connected with the control instrument, the control instrument comprises a fluorescence microscope, a MEMS pressure sensor array, a plurality of micro flow pumps and a plurality of air pumps, the MEMS pressure sensor array comprises a plurality of MEMS pressure sensors, and the micro flow pumps are all communicated with the chip body.

[0007] The fluid control channel is used for injecting fluid and dynamically adjusting the shear force waveform amplitude and frequency; the cell culture cabin is used for mixing solution to simulate pathological microenvironment to culture cell growth; and the control instrument is used for controlling the pulsatile frequency and amplitude of fluid and monitoring the shear force distribution uniformity and transient shear force change.

[0008] The principle of the basic scheme is that: the core of the present application is to realize the accurate simulation of the cell pulsatile shear force environment through the precisely designed fluid control channel and cell culture cabin and the control instrument working in cooperation therewith.

[0009] The microfluidic chip is composed of two parts of a chip body and a control instrument. The fluid control channel and the cell culture cabin are arranged on the chip body, and the two are communicated through a variable resistance structure, so that the fluid can flow smoothly therein. The main function of the fluid control channel is to inject fluid and dynamically adjust the shear force waveform amplitude and frequency through a series of complex components. These components include parallel elastic chambers, injection ports, micro flow pumps, pneumatic valves, 'Y' shaped shunt channels, spiral flow channels and resistance channel pieces, etc. When the fluid is injected into the elastic chamber, different pressures of the fluid can be generated due to the sequentially increasing size of the chamber, the fluid is collected after passing through the 'Y' shaped shunt channel, and then enters the resistance channel piece through the spiral flow channel, so that a complex fluid dynamics environment is formed in the cell culture cabin, and the pulsatile shear force suffered by the cell in the body is simulated.

[0010] The cell culture cabin is used for mixing solution to simulate pathological microenvironment, so as to provide a growth environment close to the body for the cell. It receives the fluid from the fluid control channel, so that the cell can grow in such an environment. The cell culture cabin comprises a micro column mixing cabin and a cell cabin, the mixing micro column is arranged in the micro column mixing cabin, so that the fluid can be further mixed, the uniformity of the cell culture environment is improved, and it is ensured that the shear force suffered by the cell is uniform and stable. The cell cabin is paved with a porous membrane, which can intercept the cell to prevent it from flowing away with the fluid, and is communicated with the outside through a gradient stiffness channel to simulate the mechanical environment of the cell in the body, so that the cell can grow in an environment closer to the physiological state.

[0011] The control instrument plays a crucial role in the simulation process, which includes a fluorescence microscope, a MEMS pressure sensor array, several microfluidic pumps, and several air pumps. The fluorescence microscope is used to observe the phosphorescence decay in the spiral flow channel, thereby analyzing the uniformity of shear force distribution. The MEMS pressure sensor array is used to monitor the uniformity of shear force distribution and the instantaneous shear force changes, ensuring the accuracy and stability of the shear force during the experiment. The microfluidic pumps and air pumps are used to adjust the flow rate of the fluid and the opening and closing of the pneumatic valve, respectively, thereby achieving dynamic adjustment of the shear force waveform amplitude and frequency to meet different experimental needs.

[0012] The beneficial effects of the basic scheme are: 1. The present application can more realistically simulate the microenvironment of cells in vivo, especially the pulsatile shear force environment. Traditional macroscopic experimental systems often cannot accurately reproduce the physiological fluid dynamics characteristics, while the present application realizes the accurate simulation of the pulsatile shear force environment of cells through the microfluidic technology platform, combined with precise fluid control channels and cell culture chamber design, and advanced control instruments. This has important significance for studying the biomechanical properties of cells, and helps to better understand the behavior and function of cells in vivo.

[0013] 2. The present application improves the accuracy of experimental results. Since the waveform amplitude and frequency of the shear force can be accurately controlled, and the distribution and changes of the shear force can be monitored in real time, the biological response of cells under the pulsatile shear force environment can be more accurately evaluated. This helps to reduce experimental errors and improve the reliability and repeatability of experimental results.

[0014] 3. The present application has wide application value in biomedical research. It can be used for research in tissue engineering, disease model construction, and drug screening, etc. For example, in tissue engineering, the specific mechanical environment in vivo can be simulated to promote cell proliferation and differentiation, thereby accelerating tissue regeneration and repair. In disease model construction, the mechanical environment under disease conditions can be simulated to study the mechanisms of disease occurrence and development. In drug screening, the effects of drugs on cells under specific mechanical environments can be evaluated to provide strong support for drug development.

[0015] 4. The present application promotes the research progress of cell mechanical microenvironment. By simulating the periodic pulsatile shear force environment in the vascular system, the present application provides a new experimental platform for researchers, which helps to explore the influence of cell mechanical microenvironment on cell function and its potential application in tissue engineering, regenerative medicine, etc. This will help to promote breakthroughs in the field of biomedicine and provide new ideas and strategies for future disease treatment and tissue repair.

[0016] Further, the fluid control channel includes a plurality of parallel elastic chambers, the sizes of the elastic chambers are sequentially increased, one side of each of the elastic chambers is communicated with an injection port, the injection port is communicated with a micro-flow pump output pipe, the micro-flow pump output pipe is respectively communicated with a corresponding micro-flow pump, the other side of the elastic chamber is provided with a "Y" shape shunt channel, the elastic chamber and the "Y" shape shunt channel are communicated, a pneumatic valve is arranged at the communication position, the pneumatic valve is communicated with a gas pump, one end of the "Y" shape shunt channel is communicated with a spiral flow channel, and the bottom end of the spiral flow channel is slidably communicated with a resistance channel piece.

[0017] The beneficial effect of the basic scheme is that the fluid control channel is designed by containing a plurality of parallel elastic chambers, which significantly enhances the dynamic adjustment ability of the shear force waveform. This design enables the chip to flexibly adjust the fluid of different flow and pressure, thereby generating complex and variable shear force waveforms, including pulsation and non-steady-state shear force, which helps researchers to better understand the mechanical environment of cells in vivo.

[0018] Further, the spiral flow channel is vertically arranged, and the pitch gradually decreases from top to bottom.

[0019] The beneficial effect of the basic scheme is that the vertically arranged spiral flow channel significantly optimizes the space utilization, making the structure of the entire micro-fluidic chip more compact. This layout not only reduces the area occupied by the chip, but also enables more functional components to be integrated in limited experimental space. The design of gradually decreasing pitch from top to bottom realizes the progressive adjustment of fluid flow speed, thereby generating gradually increasing shear force. During the process of fluid flow, due to the change of pitch and the change of flow channel curvature, complex vortex and turbulence are generated in the spiral flow channel, which not only increases the mixing effect of the fluid, but also realizes the uniform distribution of cells through the centrifugal effect.

[0020] Further, the inner wall of the spiral flow channel is coated with a phosphorescent nano layer, and the inner walls of the inlet and outlet of the spiral flow channel are fixedly connected with MEMS pressure sensors.

[0021] The beneficial effect of the basic scheme is that 1, the spiral flow channel is coated with a phosphorescent nano layer, which significantly enhances the visualization ability of the shear force. The phosphorescent nano layer can produce optical signal changes when subjected to shear force, and this change is related to the size and distribution of the shear force. By monitoring the attenuation area of the phosphorescent signal, researchers can directly observe the distribution of the shear force in the spiral flow channel, thereby realizing the visualization analysis of the shear force environment.

[0022] 2, the MEMS pressure sensor can monitor the pressure changes at the inlet and outlet of the spiral flow channel in real time, and then calculate the size of the shear force. This real-time monitoring method can capture the dynamic change process of the shear force, providing a powerful tool for researchers to study the response of cells under different shear force environments.

[0023] Further, the inner wall of the chip body is in sliding fit with the resistance channel member, a plurality of resistance flow channels are opened on the resistance channel member, the diameters of the resistance flow channels are all not greater than that of the spiral flow channel and are all in communication with the outlet of the spiral flow channel, the length of the resistance flow channel increases with the decrease of the diameter, the inlet and outlet of the resistance flow channel are both provided with a tapered transition cavity, and the outer wall of the resistance channel member is paved with a sealing layer.

[0024] The beneficial effects of the basic scheme are: 1. The design of the resistance channel member significantly enhances the flexibility of fluid control. By opening the variable resistance flow channels, multiple path options are provided for the flow of fluid in the microfluidic chip. Researchers can flexibly select different combinations of resistance flow channels according to experimental needs to accurately regulate the flow speed and shear force of the fluid.

[0025] 2. The design of the resistance channel member helps to improve the accuracy of shear force simulation. By accurately changing the size of the resistance flow channel, stable shear force can be ensured when the fluid passes through the resistance channel member. The tapered transition cavities at the inlet and outlet of the resistance flow channel help to reduce turbulence and vortex phenomena during fluid flow, making the fluid flow more stable, thereby improving the accuracy and reliability of shear force simulation.

[0026] 3. The design of the resistance channel member also helps to improve the repeatability and reliability of the experiment. Since the shape and size of the resistance flow channel are precisely controlled, the flow speed and shear force of the fluid can be kept consistent in each experiment. This consistency helps to reduce experimental errors and uncertainties, making the experimental results more reliable and repeatable.

[0027] Further, a plurality of variable magnetic strips parallel to the inlets of the resistance flow channels are embedded on the bottom wall of the resistance channel member, and fixed magnetic strips corresponding to the bottom wall of the resistance channel member are embedded on the inner wall of the chip body, the fixed magnetic strips being parallel to the outlet of the spiral flow channel.

[0028] The beneficial effects of the basic scheme are: 1. Since the design of the variable magnetic strips allows the experimenter to adjust their position as needed, this design enables the experimenter to easily switch the resistance flow channels and conveniently align the outlet of the spiral flow channel through the fixed magnetic strips, thereby exploring the effects of different shear force environments on cell growth and function, improving the flexibility and repeatability of the experiment.

[0029] 2. Compared with traditional mechanical positioning methods, using magnetic strips to switch resistance flow channels has the advantages of simple operation and easy maintenance, not only reducing the difficulty and cost of experimental operation, but also helping to improve experimental efficiency and safety.

[0030] Further, the cell culture chamber includes a micro-column mixing cabin, one side of the micro-column mixing cabin is communicated with the resistance flow channel, a plurality of vertical mixing micro-columns are fixedly connected to the inner wall of the micro-column mixing cabin, and a plurality of side flow channels are further communicated with the side of the micro-column mixing cabin communicated with the resistance flow channel.

[0031] The beneficial effects of the basic scheme are: 1. The design of the mixing micro-column aims to optimize the nutrient supply and metabolic waste discharge in the cell culture process by increasing the disturbance and mixing efficiency of the fluid. When the fluid passes through the micro-column mixing cabin, the mixing micro-column will guide the fluid to generate vortex and shear force. These complex flow patterns help to evenly distribute nutrients and promote the full contact between cells and nutrients, providing a more stable and uniform growth environment for cells.

[0032] 2. A plurality of side flow channels are connected to the micro-column mixing cabin, and the side flow channels are communicated with the micro-flow pump through the liquid adding port in the top wall of the chip body. The design of the side flow channel allows the experimenter to add or remove specific chemicals or culture medium to the cell culture chamber without disturbing the main culture environment, so as to realize accurate regulation of the cell culture environment and establishment of chemical gradient.

[0033] 3. The side flow channel is provided with a delay channel with a zigzag structure. This design further enhances the functionality of the cell culture chamber. The delay channel can adjust the flow speed of the fluid, so that the introduced chemicals or culture medium can be gradually and uniformly dispersed into the entire culture environment. This gradual release method helps to avoid sudden impact or pressure changes on cells, thereby ensuring the healthy growth and normal metabolism of cells

[0034] Further, the other side of the micro-column mixing cabin is communicated with a cell cabin, the bottom of the cell cabin is paved with a porous membrane, the bottom wall of the cell cabin is communicated with a gradient stiffness channel, the inner walls of the inlet and outlet of the cell cabin are fixedly connected with MEMS pressure sensors, the inner walls of the inlet and outlet of the gradient stiffness channel are fixedly connected with MEMS pressure sensors, and the gradient stiffness channel is communicated with the outside at the end.

[0035] The beneficial effects of the basic scheme are: 1. The porous membrane in the cell cabin not only provides a platform for cell attachment and growth, but also effectively controls the flow mode and speed of the fluid in the cell cabin. This design allows cells to be cultured under conditions closer to physiological conditions, thereby better simulating the growth and metabolism of cells in vivo.

[0036] 2、Gradient stiffness channel can simulate the shear force change under complex physiological environment such as lesion area. This design not only improves the simulation accuracy of the experiment, but also provides researchers with more abundant and accurate experimental data, which helps to reveal the biological behavior of cells under complex mechanical environment.

[0037] 3、MEMS pressure sensor can monitor the pressure change of fluid in microfluidic chip in real time. This design not only improves the accuracy and repeatability of the experiment, but also provides researchers with more intuitive and accurate experimental data. By collecting and analyzing these pressure data, researchers can further understand the flow state and distribution rule of fluid in microfluidic chip, thereby providing strong support for optimizing experimental design and improving experimental efficiency.

[0038] Further, the gradient stiffness channel includes a plurality of pre-strained pipes, the stiffness of the pre-strained pipes in the middle is smaller than that of the pre-strained pipes at both ends, and a spiral air duct is opened in each pre-strained pipe, the first end and the last end of the spiral air duct extend downward through the bottom wall of the chip body and are communicated with the air pump.

[0039] The beneficial effects of the basic scheme are: 1. The pre-strained pipe as the basic unit of the gradient stiffness channel fully considers the complexity of the cell mechanical microenvironment. The spiral air duct is opened in each pre-strained pipe, which not only enhances the flexibility of the pipe wall, but also allows the stiffness to be adjusted by the air pump. The design of the spiral air duct allows gas to be evenly distributed inside the pipe wall, thereby avoiding the problem of uneven stiffness change caused by uneven gas distribution.

[0040] 2. The design of the gradient stiffness channel simulates the complex mechanical environment in the vascular system, especially the change of the stiffness of the vascular wall in the pathological environment such as the lesion area. Not only provides a new technical means for cell biomechanics experiment, but also provides a more real and reliable experimental platform for scientific research in the fields of tissue engineering, disease model construction and drug screening. This helps to promote breakthrough progress in related fields, and provides new research ideas and methods for disease treatment and tissue regeneration.

[0041] A detection method for cell pulsatile shear force environment based on the above-mentioned microfluidic chip for cell pulsatile shear force environment, comprising the following steps:

[0042] Step 1: Pretreatment and cell inoculation, flush all flow channels with 75% ethanol, ultraviolet sterilization for 30 min, inject protein adsorption inhibitor, inject endothelial cell suspension through the injection port, use the centrifugal effect of the spiral flow channel to achieve uniform distribution, until the cells are all trapped by the porous membrane, close the injection port, liquid inlet and gradient stiffness channel, and place in a 37℃, 5% CO2 incubator for 48 hours until the cell fusion degree is ≥90%;

[0043] Step two, pulse shear force parameter setting, set pulse mode through computer, set elastic chamber air pressure, and manually select resistance channel combination, and also can simulate lesion area shear force through air pump regulating pre-strain pipe stiffness, set chemical gradient through side flow channel;

[0044] Step three, pulse shear force loading and monitoring, start micro flow pump, generate bidirectional pulsatile flow by cooperating with elastic chamber and pneumatic valve, use fluorescence microscope to observe and record phosphorescent signal attenuation area of helical flow channel inner wall, adjacent MEMS sensor collects pressure difference ΔP with 1 kHz sampling rate;

[0045] Step four, integrate data and verify, analyze shear force distribution uniformity according to phosphorescent signal attenuation area, calculate instantaneous shear force according to pressure difference ΔP collected by MEMS sensor, superimpose spatial distribution of optical signal and pressure time sequence data to generate three-dimensional shear force-time-space heat map, collect and detect vascular active substances secreted by cells through side flow channel 24 hours after loading, and compare with static culture group. BRIEF DESCRIPTION OF DRAWINGS

[0046] Figure 1 It is an isometric view of the microfluidic chip for cell pulsatile shear force environment in the embodiment of the application.

[0047] Figure 2 It is a side view of the microfluidic chip for cell pulsatile shear force environment in the embodiment of the application.

[0048] Figure 3 It is a side view of the microfluidic chip for cell pulsatile shear force environment in the embodiment of the application.

[0049] Figure 4 It is a bottom view of the elastic chamber in the embodiment of the application.

[0050] Figure 5 It is a bottom view of the resistance channel piece in the embodiment of the application.

[0051] Figure 6 It is a bottom view of the pre-strain pipe in the embodiment of the application.

[0052] Figure 7 It is Figure 5 It is an enlarged A part.

[0053] Figure 8 It is a schematic diagram of the detection method for cell pulsatile shear force environment in the embodiment of the application.

[0054] The reference signs in the drawings of the specification include: 1, chip body; 2, micro flow pump output pipe; 3, resistance passage; 4, air pump; 5, injection port; 6, elastic chamber; 7, pneumatic valve; 8, “Y” shape shunt passage; 9, spiral flow channel; 10, MEMS pressure sensor; 11, phosphor nano layer; 12, fixed magnetic strip; 13, variable magnetic strip; 14, resistance flow channel; 15, micro column mixing cabin; 16, mixing micro column; 17, cell cabin; 18, porous membrane; 19, gradient stiffness passage; 20, spiral airway; 21, liquid inlet; 22, conical transition cavity; 23, side flow channel; 24, delay channel. DETAILED DESCRIPTION

[0055] The following is further described in detail through specific embodiments:

[0056] Example 1

[0057] Basically as shown in the accompanying Figure 1 , Figure 2 , Figure 3 , Figure 4 , Figure 5 , Figure 6 and Figure 7 : a microfluidic chip for cell pulsatile shear force environment, comprising a chip body 1 and a control instrument, the chip body 1 is provided with fluid control channels and cell culture cabins, the fluid control channels communicate with the cell culture cabins, the chip body 1 is bolted and fixedly connected with the control instrument, the control instrument includes a fluorescence microscope, a MEMS pressure sensor 10 array, a plurality of micro flow pumps and a plurality of air pumps 4, the MEMS pressure sensor 10 array includes a plurality of MEMS pressure sensors 10, and the micro flow pumps all communicate with the chip body 1.

[0058] The control instrument is used for controlling the pulsatile frequency and amplitude of the fluid and monitoring the shear force distribution uniformity and transient shear force change.

[0059] The fluid control channel is used for injecting fluid and dynamically adjusting the shear force waveform amplitude and frequency, and comprises a plurality of elastic chambers 6 connected in parallel, the elastic chambers 6 are sequentially increased in size, and the volume ratio of the elastic chambers 6 is 1:2:4 if three elastic chambers 6 are arranged, the elastic chambers 6 are all communicated with injection ports 5 on one side, the injection ports 5 are all communicated with micro-flow pump output pipes 2, the micro-flow pump output pipes 2 are respectively communicated with corresponding micro-flow pumps, the other side of the elastic chambers 6 is provided with a “Y”-shaped shunt channel 8, the elastic chambers 6 and the “Y”-shaped shunt channel 8 are all communicated, and the communication parts are all bonded with pneumatic valves 7, the pneumatic valves 7 are all respectively communicated with corresponding air pumps 4, the response time of the pneumatic valves 7 is less than 10 ms, one end of the “Y”-shaped shunt channel 8 is communicated with a spiral flow channel 9, the bottom end of the spiral flow channel 9 is slidably communicated with a resistance channel piece 3, the spiral flow channel 9 is arranged vertically in the axial direction, the pitch gradually decreases from top to bottom, the inner wall of the spiral flow channel 9 is coated with a phosphorescent nano layer 11 with a thickness of 10 μm, and the excitation light is separated from the wavelength of the cell autofluorescence, the inner walls of the inlet and outlet of the spiral flow channel 9 are inlaidly connected with MEMS pressure sensors 10, the inner wall of the chip body 1 is in sliding fit with the resistance channel piece 3, a plurality of resistance flow channels 14 are opened on the resistance channel piece 3, the diameters of the resistance flow channels 14 are all not greater than the diameter of the spiral flow channel 9 and can all be communicated with the outlet of the spiral flow channel 9, for example, if three resistance flow channels 14 are arranged, the diameters are 20 / 50 / 100 μm respectively, and the lengths are 5 / 10 / 20 mm respectively, the lengths of the resistance flow channels 14 all increase with the decrease of the diameters (that is, the lengths of the resistance flow channels 14 are inversely proportional to the diameters), tapered transition cavities 22 are opened at the inlet and outlet of the resistance flow channels 14, a sealing layer is laid on the outer wall of the resistance channel piece 3, a plurality of variable magnetic strips 13 parallel to the inlets of the resistance flow channels 14 are inlaid on the bottom wall of the resistance channel piece 3, and a fixed magnetic strip 12 corresponding to the bottom wall of the resistance channel piece 3 is inlaid on the inner wall of the chip body 1, and the fixed magnetic strip 12 is parallel to the outlet of the spiral flow channel 9.

[0060] The cell culture chamber is used for culturing cell growth in a mixed solution simulation pathological microenvironment, and the cell culture chamber comprises a micro-column mixing cabin 15, one side of the micro-column mixing cabin 15 is communicated with a corresponding resistance flow channel 14, a plurality of vertical mixing micro-columns 16 are fixedly connected to the inner wall of the micro-column mixing cabin 15, the diameters of the mixing micro-columns 16 are all 20 μm, the distribution intervals are all 50 μm, and the side of the micro-column mixing cabin 15 communicated with the resistance channel piece 3 is further communicated with a plurality of side flow channels 23; a liquid adding opening 21 is formed in the top wall of the chip body 1, the side flow channels 23 are communicated with the micro-flow pump output pipe 2 through the liquid adding opening 21, the side flow channels 23 are all provided with a delay channel with a zigzag structure, the other side of the micro-column mixing cabin 15 is communicated with a cell cabin 17, a porous membrane with a pore size of 3 μm and a porosity of 30% is laid at the bottom of the cell cabin 17, the bottom wall of the cell cabin 17 is communicated with a gradient stiffness channel 19, the inner walls of the inlet and outlet of the cell cabin 17 are all inlaidly connected with MEMS pressure sensors 10, the inner walls of the inlet and outlet of the gradient stiffness channel 19 are all inlaidly connected with MEMS pressure sensors 10, the gradient stiffness channel 19 is communicated with the outside, the gradient stiffness channel 19 comprises a plurality of pre-strain pipes, the stiffnesses of the pre-strain pipes are different, the stiffness of the middle pre-strain pipe is smaller than that of the pre-strain pipes at two ends, and spiral air ducts 20 are all formed in the pre-strain pipes, the first end and the last end of the spiral air duct 20 are both downwardly extended through the bottom wall of the chip body 1 and are both communicated with an air pump 4.

[0061] The pre-strain pipe material is mixed with a photosensitive crosslinking agent, before the pre-strain pipe is assembled into the chip body 1, the crosslinking density is controlled by the difference of the exposure time and intensity of ultraviolet light, so that the pre-strain stiffness of the pre-strain pipe is changed.

[0062] The specific implementation process is as follows: the existing micro-fluidic chip for cell pulsating shear force environment mainly changes the shear force in a hydraulic way, but it is difficult to dynamically adjust the flow direction and frequency according to the preset waveform, not to mention integrating the chemical gradient, the substrate stiffness gradient and other microenvironment conditions with the shear force simulation experiment.

[0063] In the present application, the micro-flow pump first injects the fluid into the elastic chamber 6, the pneumatic valve 7 is periodically opened and closed according to the preset waveform, the chamber flow output is controlled, the large volume chamber (4 μL) is used for buffering the low frequency pulsation (0.1-2 Hz), the small volume chamber (1 μL) is used for high frequency pulsation (2-20 Hz), the chamber volume and the timing of the pneumatic valve 7 are adjusted, and the complex waveform (such as pulsating shear force) is dynamically generated, covering the physiological range (0.1-50 dyn / cm 2 ) of the arteriole to the capillary.

[0064] Then the fluid is collected by the "Y" shaped split channel 8 into the vertically arranged spiral flow channel 9, the pitch change leads to the increase of the flow rate gradient, and the centrifugal effect is generated, which makes the cells uniformly distributed downstream. The phosphor nano layer 11 attenuates the light intensity under the action of shear force, and the fluorescent microscope captures the attenuation area in real time, thereby inverting the shear force distribution. The uniformity of the flow rate distribution of the fluid through the spiral flow channel 9 is improved (coefficient of variation CV < 15%).

[0065] The fluid flowing through the spiral flow channel 9 enters the pre-rotated resistance flow channel 14. Since the bottom of the resistance channel piece 3 has a variable magnetic strip 13 corresponding to the fixed magnetic strip 12, whether manually or mechanically adjusting the switching resistance flow channel 14 can more easily align the outlet of the spiral flow channel 9, thereby improving the experimental efficiency and experimental repeatability. When a high resistance flow channel (20 μm) is selected, the system flow resistance increases, and the shear force amplitude increases (up to 50 dyn / cm 2 ), and the flow resistance adjustment range of the resistance channel piece 3 covers 0.1-12.8 Pa·s / μL, supporting high-precision simulation of pathological environments (such as arterial stenosis).

[0066] The mixing micro column 16 arranged in the micro column mixing cabin 15 mainly mixes the liquid added by the side flow channel 23 and the liquid of the resistance flow channel 14 through the local turbulence formed, and enhances the internal flow rate uniformity of the liquid, balancing the local impact on the subsequent cells. The delay channel of the side flow channel 23 delays the diffusion speed, helping to form a stable chemical gradient, and the spatial resolution of the chemical gradient reaches 100 μm, and the cell microenvironment heterogeneity simulation capability is improved.

[0067] The cells are trapped and constrained on the porous membrane and continuously grow, and the gradient stiffness channel 19 connected in sequence by the cell cabin 17 can use the expansion of the middle pre-strained tube with smaller stiffness to reduce the stiffness to simulate vascular lesions. The air pump 4 injects gas into the spiral air channel 20 of the pre-strained tube, and the high pressure makes the pre-strained tube reduce the stiffness, and the low pressure recovers. The middle pre-strained tube (1 MPa) simulates the softening area of the plaque, and the two ends (50 kPa) simulate the healthy blood vessel wall. The dynamic adjustment range of the base stiffness reaches 200 times, supporting the mechanical heterogeneity simulation of vascular lesions.

[0068] The MEMS pressure sensor 10 distributed at the inlet and outlet of different pipelines monitors the flow channel pressure difference (ΔP) at a sampling rate of 1 kHz, and calculates the instantaneous shear force through the following formula:

[0069]

[0070] In the formula, η is the viscosity, Q is the flow rate, and w / h / l is the chamber width / height / length.

[0071] Example 2

[0072] The difference from the above example is that, as shown in the attached Figure 1 ,Figure 2 、 Figure 3 and Figure 8 A detection method for cell pulsatile shear stress environment based on the above-mentioned microfluidic chip for cell pulsatile shear stress environment, comprising the following steps:

[0073] Step one, pretreatment and cell inoculation, rinse all flow channels with 75% ethanol, ultraviolet sterilization for 30 min, inject protein adsorption inhibitor, inject endothelial cell suspension through injection port 5, use the centrifugal effect of spiral flow channel 9 to achieve uniform distribution, until the cells are all intercepted by the porous membrane, close the injection port, liquid inlet and gradient stiffness channel, and place in a 37°C, 5% CO2 incubator for 48 hours to culture to a cell fusion degree of ≥90%;

[0074] Step two, pulse shear stress parameter setting, set the pulsatile mode by computer, the air pressure of the elastic chamber 6, and manually select the resistance channel combination, and at the same time, the shear stress of the lesion area can also be simulated by adjusting the stiffness of the pre-strain tube through the air pump 4, and the chemical gradient can be set through the side flow channel 23;

[0075] Step three, pulse shear stress loading and monitoring, start the microfluidic pump, generate bidirectional pulsatile flow in cooperation with the elastic chamber 6 and the pneumatic valve 7, use a fluorescence microscope to observe and record the phosphorescent signal attenuation area on the inner wall of the spiral flow channel 9, and adjacent MEMS sensors collect pressure difference ΔP at a sampling rate of 1 kHz;

[0076] Step four, integrate data and verify, analyze the uniformity of shear stress distribution according to the phosphorescent signal attenuation area, calculate the instantaneous shear stress according to the pressure difference ΔP collected by the MEMS sensor, superimpose the spatial distribution of the optical signal and the pressure time series data to generate a three-dimensional shear stress-time-space heat map, 24 hours after loading, collect and detect the vascular active substances secreted by the cells through the side flow channel 23, and compare with the static culture group.

[0077] The specific experimental process is as follows:

[0078] 1. Chip pretreatment: rinse all flow channels with 75% ethanol for 30 min, and inject 0.1% Pluronic F-127 protein adsorption inhibitor after ultraviolet sterilization.

[0079] Endothelial cell suspension (density 1×10 6 cells / mL) passes through the spiral flow channel 9, and the centrifugal effect makes the cells uniformly adhere to the porous membrane, and is statically cultured for 48 hours to a fusion degree of ≥90%.

[0080] 2. Shear stress parameter setting:

[0081] (1) Pulsatile mode: set the sine waveform (frequency 1.5 Hz, amplitude 25 dyn / cm 2(2) Stiffness adjustment: air pump 4 injects 30 kPa gas into the pre-strained tube in the gradient stiffness channel 19, and the stiffness drops to 20 kPa (simulating plaque region).

[0082] (2) Stiffness adjustment: air pump 4 injects 30 kPa gas into the pre-strained tube in the gradient stiffness channel 19, and the stiffness drops to 20 kPa (simulating plaque region).

[0083] (3) Chemical gradient: side channel 23 injects oxidized LDL (100 μg / mL), and a concentration gradient (0→100 μg / mL, span 5 mm) is formed in the cell chamber 17 by the delay channel.

[0084] 3, Shear loading and monitoring:

[0085] (1) Dynamic loading: micro-pump drives fluid at a flow rate of 10 μL / min, and pneumatic valve 7 is opened and closed at a frequency of 1.5 Hz to generate bidirectional pulsatile flow. When the flow is reversed, 20% of the flow is returned through the bypass, enhancing oscillatory shear (OSC).

[0086] (2) Real-time monitoring: fluorescence microscope records phosphorescence decay area (exposure time per frame 10 ms), and MEMS sensor collects pressure difference data. Shear distribution uniformity analysis (CV < 15%), and pressure data is fed back to adjust the flow rate of the micro-pump in real time.

[0087] 4, Data integration and verification:

[0088] (1) Three-dimensional heat map generation: superimpose the spatial distribution of phosphorescence signal (resolution 50 μm) and pressure time series data (1 kHz) to generate a shear-time-space heat map.

[0089] (2) Biological verification: after loading for 24 hours, collect the cell secretion liquid in the side channel 23, and detect the NO / PGI2 level by ELISA. Compared with the static culture group, the eNOS expression in the pulsatile group is increased by 3.2 times, and the TEER value is increased by 40%.

[0090] 5, Experimental results:

[0091] (1) Experimental setup parameters, as shown in Table 1 below.

[0092] Table 1, experimental parameter settings and real-time monitoring data

[0093]

[0094]

[0095] (2) Biological verification results, as shown in Table 2 below.

[0096] Table 2, biological verification results

[0097]

[0098] In the actual experiment process, the cell secretion factor of endothelial cells under the influence of pulsating shear force shows a fold increase compared with the static culture control group, which proves that the simulated pulsating shear force in the application has a corresponding influence on the function of endothelial cells, and can be used as an automatic simulation experiment of pulsating shear force.

[0099] It should be noted that, in this document, the terms such as first and second are used merely to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between such entities or operations. Moreover, the terms "include", "contain" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements not only includes those elements, but also includes other elements not explicitly listed or inherent to such process, method, article or device.

[0100] The above is only an embodiment of the application, and the common knowledge of the specific structure and characteristics in the scheme is not described in detail. The person skilled in the art knows all the common technical knowledge in the field of the application before the application date or the priority date, can know all the prior art in the field, and has the ability to apply conventional experimental means before that date. The person skilled in the art can improve and implement the scheme under the guidance of this application, and some typical known structures or known methods should not be an obstacle for the person skilled in the art to implement the application. It should be noted that, for those skilled in the art, without departing from the structure of the application, a number of modifications and improvements can be made, which should also be considered as the protection scope of the application, and these will not affect the effect and practicality of the application. The protection scope of the present application should be subject to the content of its claims, and the specific embodiments in the specification can be used to explain the content of the claims.

Claims

1. A microfluidic chip for cell pulsatile shear stress environment, comprising a chip body (1) and a control instrument, characterized in that: The chip body (1) is provided with a fluid control channel and a cell culture chamber, the fluid control channel is communicated with the cell culture chamber, the chip body (1) is fixedly connected with a control instrument, the control instrument comprises a fluorescence microscope, a MEMS pressure sensor (10) array, a plurality of micro flow pumps and a plurality of air pumps (4), the MEMS pressure sensor (10) array comprises a plurality of MEMS pressure sensors (10), and the micro flow pumps are all communicated with the chip body (1); The fluid control channel is used for injecting fluid and dynamically adjusting the shear force waveform amplitude and frequency; the cell culture chamber is used for mixing solution to simulate pathological microenvironment to culture cell growth; and the control instrument is used for controlling the pulsation frequency and amplitude of the fluid and monitoring the shear force distribution uniformity and instantaneous shear force change.

2. The microfluidic chip for cell pulsatile shear stress environment according to claim 1, wherein: The fluid control channel comprises a plurality of parallel elastic chambers (6), the sizes of the elastic chambers (6) are sequentially increased, one side of each of the elastic chambers (6) is communicated with an injection port (5), the injection port (5) is communicated with a micro flow pump output pipe (2), the micro flow pump output pipe (2) is respectively communicated with a corresponding micro flow pump, the other side of the elastic chamber (6) is provided with a ''Y''-shaped shunt channel (8), the elastic chamber (6) and the ''Y''-shaped shunt channel (8) are all communicated, a pneumatic valve (7) is arranged at the communicated position, the pneumatic valve (7) is communicated with the air pump (4), and one end of the ''Y''-shaped shunt channel (8) is communicated with a spiral flow channel (9), and the bottom end of the spiral flow channel (9) is slidably communicated with a resistance channel piece (3).

3. The microfluidic chip for cell pulsatile shear stress environment according to claim 2, wherein: The spiral flow channel (9) is vertically arranged, and the pitch gradually decreases from top to bottom.

4. The microfluidic chip for cell pulsatile shear stress environment according to claim 3, wherein: The inner wall of the spiral flow channel (9) is coated with a phosphor nano layer (11), and the inner wall of the inlet and outlet of the spiral flow channel (9) is fixedly connected with the MEMS pressure sensor (10).

5. The microfluidic chip for cell pulsatile shear stress environment according to claim 4, wherein: The inner wall of the chip body (1) is slidably matched with the resistance channel piece (3), a plurality of resistance flow channels (14) are formed in the resistance channel piece (3), the diameters of the resistance flow channels (14) are all not greater than that of the spiral flow channel (9) and are all communicated with the outlet of the spiral flow channel (9), the length of the resistance flow channel (14) increases with the decrease of the diameter, the inlet and outlet of the resistance flow channel (14) are all provided with a tapered transition cavity (22), and the outer wall of the resistance channel piece (3) is paved with a sealing layer.

6. The microfluidic chip for cell pulsatile shear stress environment according to claim 5, wherein: A plurality of variable magnetic strips (13) parallel to the inlets of the resistance flow channels (14) are embedded in the bottom wall of the resistance channel piece (3), a fixed magnetic strip (12) corresponding to the bottom wall of the resistance channel piece (3) is embedded in the inner wall of the chip body (1), and the fixed magnetic strip (12) is parallel to the outlet of the spiral flow channel (9).

7. The microfluidic chip for cell pulsatile shear stress environment according to claim 6, wherein: The cell culture chamber comprises a micro column mixing cabin (15), one side of the micro column mixing cabin (15) is communicated with the resistance flow channel (14), a plurality of vertical mixing micro columns (16) are fixedly connected to the inner wall of the micro column mixing cabin (15), and one side of the micro column mixing cabin (15) communicated with the resistance channel piece (3) is further communicated with a plurality of side flow channels (23).

8. The method of claim 7, wherein: The other side of the micro-column mixed cabin (15) is communicated with a cell cabin (17), the bottom of the cell cabin (17) is paved with a porous membrane, the bottom wall of the cell cabin (17) is communicated with a gradient stiffness channel (19), the inner walls of the inlet and outlet of the cell cabin (17) are fixedly connected with MEMS pressure sensors (10), the inner walls of the inlet and outlet of the gradient stiffness channel (19) are fixedly connected with MEMS pressure sensors (10), and the terminal end of the gradient stiffness channel (19) is communicated with the outside.

9. The microfluidic chip for cell pulsatile shear stress environment according to claim 8, wherein: The gradient stiffness channel (19) comprises a plurality of pre-strained pipes, the stiffnesses of the pre-strained pipes are different, the stiffness of the middle pre-strained pipe is smaller than that of the pre-strained pipes at two ends, and the inside of each pre-strained pipe is provided with a spiral air duct (20), the first end and the terminal end of the spiral air duct (20) extend downward through the bottom wall of the chip body (1) and are communicated with the air pump (4).

10. A detection method for cell pulsating shear force environment based on the above-mentioned microfluidic chip for cell pulsating shear force environment, characterized in that: The method comprises the following steps: Step one, pretreatment and cell inoculation: flush all flow channels with 75% ethanol, ultraviolet sterilization for 30 minutes, inject a protein adsorption inhibitor, inject endothelial cell suspension through the injection port (5), realize uniform distribution by using the centrifugal effect of the spiral flow channel (9), until the cells are all intercepted by the porous membrane, close the injection port, the liquid inlet and the gradient stiffness channel, and place in a 37°C and 5% CO2 incubator for 48 hours to culture until the cell fusion degree is greater than or equal to 90%; Step two, pulse shear force parameter setting: set the pulsation mode through the computer, the air pressure of the elastic chamber (6), and manually select the resistance channel combination, and at the same time, the stiffness of the pre-strained pipe can also be adjusted through the air pump (4) to simulate the shear force of the diseased area, and the chemical gradient is set through the side flow channel (23); Step three, pulse shear force loading and monitoring: start the micro-flow pump, generate bidirectional pulsatile flow in cooperation with the elastic chamber (6) and the pneumatic valve (7), use a fluorescence microscope to observe and record the phosphorescent signal attenuation area on the inner wall of the spiral flow channel (9), and adjacent MEMS sensors collect pressure difference ΔP at a sampling rate of 1 kHz; Step four, data integration and verification: analyze the uniformity of shear force distribution according to the phosphorescent signal attenuation area, calculate the instantaneous shear force according to the pressure difference ΔP collected by the MEMS sensor, superimpose the spatial distribution of the optical signal and the pressure time sequence data to generate a three-dimensional shear force-time-space heat map, collect and detect the vascular active substances secreted by the cells through the side flow channel (23) 24 hours after loading, and compare with the static culture group.

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