CN116731857A - Organ chip for air pressure stimulation movement and cell stretching method - Google Patents
Organ chip for air pressure stimulation movement and cell stretching method Download PDFInfo
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Abstract
The invention belongs to the technical fields of biomedical engineering and microfluidics, and particularly discloses an organ chip for air pressure stimulation movement and a cell stretching method, wherein the organ chip comprises an upper runner layer and a lower runner layer, a thin film layer is arranged between the upper runner layer and the lower runner layer, a cell channel is arranged on the upper runner layer, a diversion hole communicated with the cell channel is also arranged on the lower runner layer, a fourth runner communicated with the air channel is arranged on the lower runner layer, the fourth runner is in contact with the thin film layer, and the fourth runner is of a groove structure connected end to end; the fourth flow passage is communicated with a gas passage, and one end of the gas passage, which is far away from the fourth flow passage, is connected with an injection pump system; the invention provides an organ chip capable of performing pneumatic stimulation motion of pulling motion in the horizontal direction and a cell stretching method, so that cells in a cavity can be subjected to continuous, safe and stable pulling stimulation.
Description
Technical Field
The invention belongs to the technical fields of biomedical engineering and microfluidics, and particularly relates to an organ chip for air pressure stimulation movement and a cell stretching method.
Background
Moderate exercise has a beneficial effect on the human body and the immune system of the human is enhanced. At present, macroscopic effects on the body after human exercise have been demonstrated, but microscopic cellular changes have been explored to a lesser extent. To address some of these problems and to provide a surrogate tool for the preclinical stage, early "cell culture analogs" were specifically designed to culture animal cells in an attached chamber that was perfused with recycled tissue culture medium or "blood substitutes". After these models, a "heart lung micro-machine" has emerged to combine the lung cell culture model with a heart device.
Conventional two-dimensional cell culture is difficult to construct into complex three-dimensional structures, and although animal models have contributed significantly to understanding physiology and disease and development of new drugs, researchers have long appreciated that there is often an inconsistency between animal studies and human studies, and thus a more specific modeling and testing platform for human responses is required. In recent years, organ chips have been vigorously developed as a technique for three-dimensionally culturing cells outside a human body to simulate organ functions in the human body. The dramatic expansion of the organ-chip field is achieved by the fusion of a number of previous, disparate technologies including induced pluripotent stem cells and mixed cell culture capabilities, genome editing, 3D printing, complex cell sensors, microfluidics, and micromachining engineering. In recent years, organ chips have been rapidly developed in high-throughput drug screening, drug absorption and metabolism, drug development, human circulatory system, drug toxicology, artificial bionic microenvironment, intercellular interactions, interactions of cells with extracellular matrices, novel in vitro culture platforms, and the like.
The organ chip based on the movement mode is mainly electric stimulation, and the basic structure mainly comprises a tissue chamber with cell hydrogel filled in the middle, and electrodes are added on two sides of the tissue chamber and used for providing electric stimulation for myocardial cells in the chamber. However, this structure is only used for culturing myocardial cells, and is not suitable for cell culture of other organs. Moreover, the response on the cells is not only an electrical stimulus, but also a stress stimulus, which is an important way. However, most of the pressure stimulation chips are implanted with micro air pumps, the operation and the preparation are complex, the popularization is not easy, and the existing pressure stimulation chips are mostly force for vertically compressing cells, so that the damage to the cells is easy to cause. Therefore, a new, simple strategy is needed to construct an organ-chip that can "move" in the horizontal direction, thereby allowing cells in the chamber to get continuous and safe and stable pulling stimuli.
Disclosure of Invention
The invention aims to provide an organ chip capable of performing pneumatic stimulation motion of pulling motion in the horizontal direction and a cell stretching method, so that cells in a cavity can be subjected to continuous and safe and stable pulling stimulation.
Based on the above purpose, the invention adopts the following technical scheme:
the organ chip comprises an upper runner layer and a lower runner layer, wherein a film layer is arranged between the upper runner layer and the lower runner layer, a cell channel is arranged on the upper runner layer, a diversion hole communicated with the cell channel is also arranged on the lower runner layer, a fourth runner is arranged on the lower runner layer and is in contact with the film layer, and the fourth runner is of a closed groove structure connected end to end; a gas channel communicated with the fourth flow channel is arranged in the organ chip for the air pressure stimulation movement, and one end of the gas channel, which is far away from the fourth flow channel, is connected with an injection pump system.
Further, the film layer is bonded with the lower runner layer and is in sealing connection, and the fourth runner and the film layer are made of elastic materials.
Further, the gas channel comprises a gas storage groove arranged on the lower runner layer, one end of the gas storage groove is communicated with the fourth runner, and the other end of the gas storage groove is communicated with the outside of the organ chip for the air pressure stimulation movement.
Further, the gas channel comprises a third flow channel arranged on the lower flow channel layer, one end of the third flow channel is communicated with the gas storage groove, and the other end of the third flow channel is communicated with the fourth flow channel.
Further, the gas channel comprises vent holes formed in the film layer and the upper flow channel layer, and the vent holes are communicated with the gas storage groove.
Further, a cell channel is arranged on the upper flow channel layer, the cell channel is of a groove structure, the cell channel is in contact with the film layer, and the orthographic projection of the fourth flow channel on the upper flow channel layer is positioned in the cell channel.
Further, a first runner and a second runner are arranged on the upper runner layer, and the first runner and the second runner are respectively connected with two ends of the fourth runner; the upper flow channel layer is also provided with a first flow guide hole and a second flow guide hole, one end of the first flow guide hole is communicated with the first flow channel, the other end of the first flow guide hole is communicated with the outside of the organ chip for air pressure stimulation movement, one end of the second flow guide hole is communicated with the second flow channel, and the other end of the second flow guide hole is communicated with the outside of the organ chip for air pressure stimulation movement; the first diversion holes and the second diversion holes are exposed on the outer surface of the organ chip for air pressure stimulating movement.
Further, the widths of the cross sections of the third flow channel and the fourth flow channel are consistent; the joint of the third runner and the fourth runner is a round angle; the depth of the air storage groove is consistent with that of the third flow passage and the fourth flow passage.
Further, the thickness of the upper runner layer and the lower runner layer is 1-3 mm; the thickness of the film layer is 0.05-0.1 mm.
Further, the film layer is made of PDMS.
Further, the diameter of the air storage groove is more than 100 mu m and less than 2mm.
Further, the first diversion holes are at least two, and the second diversion holes are at least two.
Further, the ratio of the width of the cell channel to the width of the fourth flow channel is 1:0.02-1:0.1; the ratio of the width of the cell channel to the width of the closed groove formed by the fourth flow channel is 1:0.5-1:0.8.
The preparation method of the organ chip for the pneumatic stimulation movement comprises the following steps,
and step 1, drawing a micro-channel structure of the organ chip, and preparing a mask by using etching equipment.
And 2, manufacturing a silicon wafer die by using the mask.
Step 3, uniformly mixing the polydimethylsiloxane prepolymer and the curing agent in a mass ratio of 10:1, pouring the mixture into a culture dish provided with a silicon wafer die, vacuum degassing, heating and curing, and stripping the polydimethylsiloxane prepolymer and the curing agent from a silicon wafer template after natural cooling to obtain an upper runner layer and a lower runner layer; and punching a first diversion hole, a second diversion hole and an upper positioning groove on the upper runner layer by using a puncher, and punching a lower positioning groove corresponding to the upper positioning groove on the lower runner layer.
And 4, uniformly mixing the polydimethylsiloxane prepolymer and the curing agent in a mass ratio of 10:1, pouring the mixture on a culture dish after vacuum degassing, enabling the bottom surface of the culture dish to face upwards, and obtaining a film layer with the thickness of 50-100 mu m after spin coating and drying.
And 5, placing the upper runner layer and the film layer together in a plasma treatment instrument for 100s, taking out the upper runner layer and the film layer after treatment, bonding the upper runner layer and the film layer within 10s, then punching vent holes, placing the upper runner layer and the film layer together with the lower runner layer in the plasma treatment instrument for 100s, bonding the upper runner layer and the film layer with the lower runner layer after taking out, and finally placing the upper runner layer and the film layer in a hot plate for drying and curing.
A cell stretching method of an organ chip using the above-mentioned pneumatic stimulation exercise comprises the following steps,
step 1, modifying a sterilized cell channel by using fibronectin, then injecting a cell suspension into the cell channel, then placing an organ chip for air pressure stimulation movement into a cell incubator, growing cells by clinging to a film layer, and finally filling the cell channel with a culture solution;
step 2, starting the injection pump system to continuously exhaust and inflate, continuously stretching the fourth runner, stretching the fourth runner to drive the film layer to stretch together with the fourth runner, and continuously pulling and stimulating cells in the transverse direction and the longitudinal direction by the film layer;
step 3, observing the cells on the thin film layer.
Further, in step 3, cells on the thin film layer corresponding to the center position of the area surrounded by the fourth flow channel are selected for observation.
Further, in step 1, the cell suspension is one of a cell suspension of human cardiac myocytes or a cell suspension of a mixture of human umbilical vein endothelial cells and human fibroblasts.
Compared with the prior art, the invention has the following beneficial effects:
according to the invention, proper flow and flow rate are adopted to continuously exhaust and charge air into the fourth flow channel through the gas channel, so that the fourth flow channel is elastically deformed, and the film layer and the fourth flow channel are driven to deform together; the fourth flow channel is arranged to be in a head-to-tail connection structure, so that the central position of a closed area formed by the fourth flow channel only generates a pulling force parallel to the film layer, the generation of a pressure perpendicular to the film layer is avoided, and the film layer obtains regular longitudinal and transverse stretching motions; compared with the existing vertical pressure stimulation cells, the invention can realize continuous and uniform pulling stimulation on cells growing on the film layer, and realize effective continuous movement on the premise of not damaging the cells.
The third flow channel is arranged between the air storage groove and the fourth flow channel, so that the influence on the central area of the fourth flow channel caused by the direct connection of the air storage groove and the fourth flow channel can be avoided. The air vents are formed in the film layer and the upper runner layer, so that the air pump and the liquid reservoir can be connected to the organ chip from the upper runner layer, and the operation is convenient. The cell is located at the position corresponding to the cell channel on the film layer, and the orthographic projection of the fourth runner on the upper runner layer is located in the cell channel, so that the influence on the cell on the film layer can be guaranteed. The first diversion hole and the second diversion hole can be connected with a liquid storage device, and continuous fluid filling can be realized by injecting different volumes of liquid into the liquid storage device, so that cells can obtain continuous fluid shearing force during growth.
Drawings
FIG. 1 is a schematic diagram of an explosion structure of embodiment 1 of the present invention;
FIG. 2 is a schematic view showing the structure of the lower surface of the upper runner layer according to embodiment 1 of the present invention;
FIG. 3 is a schematic diagram showing the upper surface structure of the upper runner layer according to embodiment 1 of the present invention;
FIG. 4 is a schematic view of a thin film layer according to example 1 of the present invention;
FIG. 5 is a schematic diagram showing the upper surface structure of the lower runner layer according to embodiment 1 of the present invention;
FIG. 6 is an enlarged view of a portion of FIG. 5;
FIG. 7 is a schematic diagram showing the recovery state of the pneumatic stimulated stretching exercise according to example 1 of the present invention;
FIG. 8 is a drawing showing the state of stretching motion of the pneumatic pressure-stimulated stretching motion of example 1 of the present invention;
FIG. 9 is a schematic diagram showing the connection of a gas moving organ chip and a liquid reservoir according to embodiment 2 of the invention;
FIG. 10 is a schematic diagram showing the connection of a gas moving organ chip and a syringe pump system according to embodiment 3 of the invention;
FIG. 11 is a schematic diagram of a cell stretched during the air extraction process according to example 3 of the present invention;
FIG. 12 is a schematic diagram of the cell stretching process in example 3 of the present invention;
FIG. 13 is a schematic diagram showing the growth and arrangement of cells before pneumatic stimulation in example 3 of the present invention;
FIG. 14 is a schematic representation of cell growth and alignment after pneumatic stimulation in example 3 of the present invention.
In the figure: the upper flow channel layer 1, the film layer 2, the lower flow channel layer 3, the liquid reservoir 4, the first flow channel 11, the second flow channel 12, the cell channel 13, the upper vent 14, the first deflector hole 15, the second deflector hole 16, the upper first positioning groove 17, the upper second positioning groove 18, the lower vent 21, the gas storage groove 31, the third flow channel 32, the fourth flow channel 33, the lower first positioning groove 34, and the lower second positioning groove 35.
Detailed Description
Example 1
1-6, the organ chip for air pressure stimulation movement comprises an upper runner layer 1, a film layer 2 and a lower runner layer 3 which are sequentially arranged from top to bottom, wherein the upper runner layer 1, the film layer 2 and the lower runner layer 3 are horizontally arranged, and the upper runner layer 1, the film layer 2 and the lower runner layer 3 are relatively fixedly arranged; the upper runner layer 1 is laminated and sealed with the film layer 2, the film layer 2 is laminated and sealed with the lower runner layer 3, and the lower surface of the upper runner layer 1 is closely contacted with and flush with the film layer 2. As shown in fig. 5-6, the organ chip for air pressure stimulation movement further comprises an air channel, a fourth flow channel 33 communicated with the air channel is arranged on the upper surface of the lower flow channel layer 3, the fourth flow channel 33 is in contact with the lower surface of the film layer 2, and the fourth flow channel 33 is in a rectangular or annular groove structure connected end to end; the fourth runner 33 and the film layer 2 are both made of elastic materials; the end of the gas channel remote from the fourth flow channel 33 is connected to a syringe pump system.
As shown in fig. 2, the gas channel includes a gas storage groove 31 formed on the upper surface of the lower flow channel layer 3, and further includes a third flow channel 32 formed between the gas storage groove 31 and the fourth flow channel 33, the gas storage groove 31 has a cylindrical structure with an axis perpendicular to the lower flow channel layer 3, the third flow channel 32 has a straight groove structure perpendicular to one rectangular side of the fourth flow channel 33, one end of the third flow channel 32 is communicated with the gas storage groove 31, and the other end is communicated with the fourth flow channel 33.
As shown in fig. 2 to 4, the gas channel includes vent holes formed in the film layer 2 and the upper flow channel layer 1, the vent holes in the film layer 2 are lower vent holes 21, the vent holes in the upper flow channel layer 1 are upper vent holes 14, the upper vent holes 14 and the lower vent holes 21 penetrate and reach the gas storage groove 31, and the upper vent holes 14, the lower vent holes 21 and the gas storage groove 31 are coaxially arranged and have the same aperture.
The cell channel 13 is arranged on the lower surface of the upper runner layer 1, the cell channel 13 is of a rectangular groove structure, the cell channel 13 is in contact with the upper surface of the film layer 2, the orthographic projection of the fourth runner 33 on the horizontal plane is positioned in the orthographic projection of the cell channel 13 on the horizontal plane, and the fourth runner is completely arranged in the cell channel 13 in the overlook view.
The lower surface of the upper runner layer 1 is provided with a first runner 11 and a second runner 12 which are in a long groove shape, and the first runner 11 and the second runner 12 are respectively connected with two ends of a fourth runner 33; the upper flow channel layer 1 is also provided with a first diversion hole 15 and a second diversion hole 16 which are centrosymmetric, the aperture of the first diversion hole 15 is the same as that of the second diversion hole 16, the bottom end of the first diversion hole 15 is communicated with one end of the first flow channel 11 far away from the cell channel 13, the top end of the first diversion hole 15 is positioned on the upper surface of the upper flow channel layer 1, the bottom end of the second diversion hole 16 is communicated with one end of the second flow channel 12 far away from the cell channel 13, and the top end of the second diversion hole 16 is positioned on the upper surface of the upper flow channel layer 1. The lower surface of the upper runner layer 1 is provided with a liquid collecting tank, and the tank bottom of the liquid collecting tank is also provided with a first diversion hole 15 and a second diversion hole 16 which penetrate through the upper runner layer 1.
The width of the cross section of the third flow channel 32 and the fourth flow channel 33 is 200um, the joint of the third flow channel 32 and the fourth flow channel 33 is a round angle with the radius of 0.1-0.2 mm, and the depth of the air storage groove 31 is consistent with the depth of the third flow channel 32 and the depth of the fourth flow channel 33; the thickness of the upper runner layer 1 is consistent with that of the lower runner layer 3, the thickness of the lower runner layer 3 is 1-3 mm, the thickness of the thin film layer 2 is 0.05-0.1 mm, and the thin film layer 2 is made of PDMS.
As shown in fig. 2 and 5, the upper runner layer 1 is provided with a cross-shaped upper layer first positioning groove 17 and an upper layer second positioning groove 18, and the lower runner layer 3 is provided with a cross-shaped lower layer first positioning groove 34 and a lower layer second positioning groove 35; the upper first positioning groove 17 and the lower first positioning groove 34 overlap each other in the plan view, and the upper second positioning groove 18 and the lower second positioning groove 35 overlap each other in the plan view.
The upper vent hole 14 of the organ chip which is stimulated to move by air pressure is connected into an injection pump system, and under the condition that the injection flow rate of injection air is 500 mu l (unit of an injection pump), the deformation of the film layer 2 is measured, and the result is that the deformation amplitude of the elastic film after negative pressure stretching is 15% compared with that before stretching, and the deformation after 72 hours stretching can be immediately recovered after removing the negative pressure, so that the requirement of in-vivo cell experiments when simulating human movement can be completely met.
Example 2
The method for preparing the organ-chip for pneumatic stimulation exercise of example 1, comprising the steps of,
step 1, manufacturing a mask: according to specific experimental requirements, a micro-channel structure of the organ chip is designed and drawn by adopting computer aided design (Computer Aided Design, CAD) software, and a mask plate is prepared by using etching equipment.
Step 2, manufacturing a silicon wafer die: taking an ultrathin silicon wafer with the diameter of 5mm and the thickness of 200 mu m, soaking the ultrathin silicon wafer in Piranha solution (mixed solution of concentrated sulfuric acid and hydrogen peroxide) for 1h, cleaning the ultrathin silicon wafer with ultrapure water for 3 times, and drying the ultrathin silicon wafer on a heat release plate (the temperature is 60 ℃ for 10 min) for later use; spin-coating SU-8 2050 photoresist on a silicon wafer subjected to hydrophobization treatment to form a film layer with the thickness of about 100 mu m; placing the silicon wafer on a hot plate (the temperature is 65 ℃ and 95 ℃) for pre-baking; then placing the pre-baked silicon wafer on a photoetching platform, enabling a plane with photoresist to face upwards, horizontally placing the silicon wafer on a mask plate with a microstructure, and then adsorbing and exposing the silicon wafer with ultraviolet; then placing the exposed silicon wafer on a hot plate (the temperature is 65 ℃ and 95 ℃) for post-baking; soaking the silicon wafer in SU-8 2050 developing solution for developing to remove the photoresist of the unexposed part; and washing with isopropanol, and finally drying on a hot plate (the temperature is 220 ℃) to obtain the silicon wafer mold with the microprotrusion structure.
Step 3, manufacturing an organ chip micro-channel; uniformly mixing a proper amount of Polydimethylsiloxane (PDMS) prepolymer and a curing agent in a mass ratio of 10:1, pouring into a culture dish provided with the silicon wafer mold, vacuum degassing, heating and curing for 3 hours on a hot plate (temperature 60 ℃), and peeling the PDMS containing the micro-pit structure from the silicon wafer mold after natural cooling to obtain an upper runner layer 1 and a lower runner layer 3; then, the first deflector hole 15, the second deflector hole 16, the upper first positioning groove 17 and the upper second positioning groove 18 are punched on the upper runner layer 1 by using a puncher, and the lower first positioning groove 34 and the lower second positioning groove 35 are punched on the lower runner layer 3 for positioning.
And 4, uniformly mixing a proper amount of Polydimethylsiloxane (PDMS) prepolymer and a curing agent in a mass ratio of 10:1, pouring the mixture on a culture dish with the diameter of 10mm after vacuum degassing, enabling the bottom surface of the culture dish to face upwards, and obtaining a PDMS elastic film with the thickness of 50-100 mu m after spin coating and drying (the temperature of a hot plate is 60 ℃ for 2 hours) to obtain the film layer 2.
And 5, placing the PDMS upper runner layer 1 with the micro-pit structure and a culture dish with an elastic membrane (a film layer 2) in a plasma treatment instrument for 100s, immediately taking out after treatment, finishing bonding within 10s, punching corresponding positions of an upper vent hole 14 and a lower vent hole 21, placing the upper runner layer 1 and the film layer 2 and the lower runner layer 3 in the plasma treatment instrument for 100s, taking out, bonding again, then placing in a hot plate (the temperature is 60 ℃ for 10 min), drying and solidifying to obtain an assembled PDMS organ chip, and adhering glass tubes on the first guide hole 15 and the second guide hole 16 as a liquid reservoir 4 for standby, and pouring culture liquid into a cell channel by gravity self-driving.
Example 3
This example is a cell stretching method using the organ-chip of the pneumatic stimulation exercise of example 1, as shown in FIGS. 10 to 14, comprising the steps of,
step 1, inoculating cells; firstly, sterilizing an organ chip (hereinafter referred to as an organ chip) subjected to air pressure stimulation movement, wherein the sterilization sequence is as follows: firstly, putting a breathable box with an organ chip into a sterilizing pot, and setting the temperature to 210 ℃ for 2 hours; sterilizing the organ chip at high temperature, placing in an oven, setting the temperature at 60 ℃ for 5 hours, drying, and placing in an ultra-clean bench for irradiation with ultraviolet rays for 30 minutes. Resuscitates heart myocardial cells (AC 16) of human beings in a culture dish with the diameter of 100mm, after 90% of the cells are grown in the culture dish, the cells are removed by a pancreatin solution, supernatant is extracted after centrifugation to prepare cell suspension with the cell density of 0.75X106/ml, 10 mu l of fibronectin is firstly used for modifying the cell channel 13 of the sterilized organ chip to promote the cell wall adhesion growth, then the cell suspension is injected into the cell channel 13 through a first deflector hole 15, the organ chip is placed in a cell culture box with the temperature of 37 ℃ and the carbon dioxide concentration of 5% for 6 hours, then the cells are closely adhered to the membrane layer 2 for growth, 1000 mu l of AC16 culture solution and 400 mu l of AC16 culture solution are respectively injected into two liquid reservoirs 4, and the cell channel 13 is perfused at the moment under the action of gravity.
Step 2, cell stretching and observation; as shown in FIG. 10, the organ chip added with the AC16 culture solution is placed in an incubator, a pipeline connected with an external injection pump system is connected with an upper vent hole 14, as shown in FIGS. 11-12, the injection pump system is started, a 2ml needle tube is adopted, the flow is set to be 500 mu l, the one-way feeding time is 6s, the operation mode is continuous, the operation direction is that the operation is that the cells on the film layer 2 are firstly pumped and then pushed, periodic stretching stimulation is carried out on the cells, and the organ chip can be taken out from the incubator and placed under a microscope for photographing during observation.
The results of the cell cycle stretching exercise experiment are as follows: figures 13 and 14 show the number and morphology change of AC16 cells before and after stretching when the stretching deformation is 15%, and it can be seen from the figures that the number of cells before stretching is small and the arrangement is irregular, the number of cells after stretching is increased and the cells grow next to the stretched edge, and the two regular arrangement modes of transverse and vertical are presented, which show that the periodic stretching movement has an important influence on the proliferation and arrangement of cells.
Example 4
Other portions of this embodiment are the same as embodiment 3 except that: human Umbilical Vein Endothelial Cells (HUVECs) and human fibroblasts (NHLF) were used in place of human cardiac cardiomyocytes. Human Umbilical Vein Endothelial Cells (HUVEC) and human fibroblasts (NHLF) are mixed and inoculated into an organ chip, according to the different simulated vascular physiological environments, the flow of an injection pump system is set to be 300-500 mu l, the stretching frequency is set to be 0.08-1Hz, the culturing time is 7 days, and as a result, the human microvasculature is found to be thickened under the periodic stretching movement and easy to sprout along the stretching direction.
Claims (10)
1. The organ chip for the air pressure stimulation movement comprises an upper runner layer and a lower runner layer, wherein a film layer is arranged between the upper runner layer and the lower runner layer, a cell channel is arranged on the upper runner layer, and a diversion hole communicated with the cell channel is also arranged on the upper runner layer; the fourth runner is communicated with a gas channel, and one end, far away from the fourth runner, of the gas channel is connected with an injection pump system.
2. The baro-stimulated motion organ chip according to claim 1, wherein the film layer and the lower flow channel layer are bonded and hermetically connected, and the fourth flow channel and the film layer are made of elastic materials.
3. The air pressure-stimulated motion organ-chip of claim 2, wherein the air channel comprises an air storage groove formed on the lower flow channel layer, one end of the air storage groove is communicated with the fourth flow channel, and the other end of the air storage groove is communicated with the outside.
4. The air pressure stimulating exercise organ chip of claim 3, wherein the air channel comprises a third flow channel which is arranged on the lower flow channel layer, one end of the third flow channel is communicated with the air storage groove, and the other end of the third flow channel is communicated with the fourth flow channel.
5. The air pressure-stimulated motion organ-chip of claim 3 or 4, wherein the air channel comprises vent holes formed in the film layer and the upper flow channel layer, and the vent holes are communicated with the air storage groove.
6. The baro-stimulatory motor of claim 5, wherein the upper flow channel layer has a cell channel, the cell channel is in a groove structure, the cell channel is in contact with the thin film layer, and the orthographic projection of the fourth flow channel on the upper flow channel layer is located in the cell channel.
7. The baro-stimulated motion organ-chip according to claim 3, wherein the third flow channel and the fourth flow channel have a cross-sectional width that is uniform; the joint of the third flow channel and the fourth flow channel is a round angle; the depth of the air storage groove is consistent with that of the third flow passage and the fourth flow passage; the thickness of the upper runner layer and the lower runner layer is 1-3 mm; the thickness of the film layer is 0.05-0.1 mm.
8. A cell stretching method using the air pressure-stimulated exercise organ-chip of any one of claims 1 to 7, comprising the steps of,
step 1, modifying a sterilized cell channel by using fibronectin, then injecting a cell suspension into the cell channel, then placing an organ chip for air pressure stimulation movement into a cell incubator, growing cells by clinging to a film layer, and finally filling the cell channel with a culture solution;
step 2, starting the injection pump system to continuously exhaust and inflate, continuously stretching the fourth runner, stretching the fourth runner to drive the film layer to stretch together with the fourth runner, and continuously pulling and stimulating cells in the transverse direction and the longitudinal direction by the film layer;
step 3, observing the cells on the thin film layer.
9. The cell stretching method according to claim 8, wherein in step 3, the cells on the film layer corresponding to the center position of the area surrounded by the fourth flow channel are selected for observation.
10. The cell stretching method according to claim 8, wherein in step 1, the cell suspension is one or two of a human heart cardiomyocyte suspension, a human umbilical vein endothelial cell suspension and a human fibroblast suspension.
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