A microfluidic chip for real-time monitoring of cell interaction processes and method of use

Abstract

The application discloses a micro-fluidic chip for real-time monitoring of cell interaction process and a use method thereof. The micro-fluidic chip comprises a cover glass, a PDMS film and a bottom plate glass which are combined from top to bottom. The center of the PDMS film is provided with a first cell culture chamber and a second cell culture chamber. A plurality of fresh culture medium pools and a plurality of waste culture medium pools are arranged on the left and right sides of the cell culture chambers. The steps for real-time monitoring of cell interaction process by using the micro-fluidic chip comprise the following steps: (1) solid-state culture gel and cell A perfusion; (2) liquid-state culture gel and cell B perfusion; and (3) chip cell interaction real-time monitoring. The micro-fluidic chip has a clear solid-liquid gel interface area, can vertically record and observe cell migration behavior, is simple to operate, has clear images, and can independently complete various fluorescence signal detections on the chip.

Description

Technical Field

[0001] This invention relates to the field of real-time cell interaction monitoring technology, specifically to a microfluidic chip for real-time monitoring of cell interaction processes and its usage method. Background Technology

[0002] Three-dimensional cell culture provides an environment that more closely resembles the physiological state of cells in vivo. It has seen widespread development and application in drug screening, tumor research, stem cell differentiation, and organoid studies. Compared to traditional two-dimensional culture systems, three-dimensional cell culture more closely resembles the growth state of cells in vivo. Therefore, monitoring and evaluating cell-cell interactions during co-culture, including various behaviors such as chemotaxis, recognition, and penetration, as well as related matrix processes, is more conducive to maintaining cell phenotypes and simulating the cell microenvironment. The research results can more realistically reflect in vivo conditions, which is of great significance. In vivo tissues and organs are composed of various cells, and there are very complex interaction mechanisms between cells and their microenvironment. Focusing on observing cell morphological changes, growth processes, migration, and proliferation behaviors under single or multiple stimuli is crucial. Microenvironmental stimulation activates cell surface receptors, activating a series of intracellular signal transduction pathways and gene transcription, which in turn leads to changes in cell morphology and location through multiple stages such as alterations in cell polarity, cell adhesion and deadhesion, and cytoskeleton rearrangement.

[0003] The current standard three-dimensional approach is the Trans-well method, which involves culturing different cell types separately on the top and bottom sides of a polycarbonate membrane within a well plate, thereby achieving non-contact co-culture and induced migration studies of cells. Conventional well plates and chambers make it difficult to construct an interface transition from a two-dimensional plane to a three-dimensional space in vitro, and the vertical distribution of cells during seeding is not conducive to observation and image acquisition.

[0004] Current methods for monitoring invasion at collagen interfaces using microarrays all involve linear interfaces, making it difficult to simulate and reproduce the aforementioned three-dimensional cell spheroid migration process. Microfluidic cell chip technology has demonstrated its unique advantages in the biomedical field. By specifically designing chips to monitor the dynamics of three-dimensional cell spheroid migration and observe the migration of three-dimensional cell spheroids from a two-dimensional plane to the three-dimensional matrix, it has become an important tool and platform for cell research.

[0005] Patent CN 107955784 A discloses a method for monitoring three-dimensional cell spheroid migration based on microfluidic chip technology. The microfluidic chip is formed by bonding and sealing two PDMS membranes. The microfluidic chip mainly consists of a cell inlet pool, a collagen inlet pool, a culture medium inlet pool, a waste liquid pool, a cell culture chamber, a culture medium perfusion chamber, a cell spheroid capture tank, and a cell migration chamber. The method mainly includes the following steps: (1) chip collagen perfusion; (2) chip cell spheroid inoculation and culture; (3) real-time monitoring of chip cell migration. However, this invention adds the collagen working solution and cell suspension separately, which is inconvenient to operate. The PDMS membrane has poor light transmittance, resulting in unclear images during observation. The collagen is solid during culture, making it difficult to obtain clear images when calculating the area of ​​three-dimensional cell spheroid migration into the collagen and the distance the cells move.

[0006] Therefore, in order to study the complex interaction mechanisms between cells and between cells and the extracellular matrix in a gravitational microenvironment, developing a microfluidic chip that is suitable for cell growth in three dimensions and can be used to continuously monitor cell migration behavior can provide a more reliable and convenient tool for clinical research. Summary of the Invention

[0007] In view of the above-mentioned prior art, the purpose of this invention is to provide a microfluidic chip for real-time monitoring of cell interaction processes and a method for using it. This microfluidic chip is simple to operate, has no background fluorescence, excellent light transmittance, a clear solid-liquid gel interface region, and can vertically record and observe cell migration behavior.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] The first aspect of the present invention provides a microfluidic chip for real-time monitoring of cell interaction processes, comprising a PDMS membrane, wherein a cell culture chamber is provided at the center of the PDMS membrane, the cell culture chamber being divided into a first cell culture chamber and a second cell culture chamber; one side of the first cell culture chamber is connected to a first fresh culture medium pool, and the opposite side of the first cell culture chamber is connected to a first waste culture medium pool; the opposite sides of the second cell culture chamber are respectively connected to a second waste culture medium pool, and the bottom of the second cell culture chamber is connected to a second fresh culture medium pool.

[0010] Preferably, the bottom of the second cell culture chamber is connected to the second fresh culture medium pool via a culture medium input channel. The culture medium input channel is divided into a total culture medium input channel and several sub-culture medium input channels. The sub-culture medium input channels are connected to the bottom of the second cell culture chamber, and the two ends of the total culture medium input channel are respectively connected to the second fresh culture medium pool.

[0011] Preferably, the first fresh culture medium tank, the second fresh culture medium tank, and the second waste culture medium tank are in a straight line, and the second fresh culture medium tank, the first waste culture medium tank, and the second waste culture medium tank are in a straight line.

[0012] Preferably, the cell culture chamber is topped with a cover glass plate and the PDMS membrane is bottomed with a base glass plate.

[0013] More preferably, the cover glass is quartz glass, and the cover glass, PDMS film, and base glass are all rectangular. The thickness of the cover glass and base glass is 0.5-1.5 mm, the thickness of the PDMS film is 1.5-2.5 mm, the PDMS film and base glass are the same size, the width of the cover glass is less than or equal to the width of the PDMS film, and the length of the cover glass is less than the distance between the second waste culture medium pools.

[0014] Preferably, the cell culture chamber occupies 1 / 4 to 1 / 2 of the PDMS membrane area. The cell culture chamber is divided into a first cell culture chamber and a second cell culture chamber by a movable partition. The first cell culture chamber and the second cell culture chamber are rectangular and the same size.

[0015] The second aspect of the present invention provides the application of the above-mentioned microfluidic chip in real-time monitoring of cell interaction processes.

[0016] A third aspect of the present invention provides a method for real-time monitoring of cell interaction processes using the aforementioned microfluidic chip, comprising the following steps:

[0017] (1) Solid culture gel and perfusion of cell A

[0018] The PDMS membrane was attached to the base glass. The working solution of gel A and the suspension of cell A were mixed evenly and injected into the second cell culture chamber until it was full. Gel A was cured. After curing, the partition was removed and the cover glass was placed above the cell culture chamber to culture the microfluidic chip.

[0019] (2) Liquid culture gel and perfusion of cells B

[0020] After mixing gel B working solution and cell B suspension evenly, inject the mixture into the first cell culture chamber until it is full, and then culture the microfluidic chip.

[0021] (3) Real-time monitoring of cell-chip interactions

[0022] Inject culture medium A into the second cell culture chamber through the second fresh medium reservoir, inject culture medium B into the first cell culture chamber through the first fresh medium reservoir, and allow the waste culture medium to flow out through the waste medium reservoir for collection; place the microfluidic chip upright in a live cell workstation, monitor the interface between the first cell culture chamber and the second cell culture chamber, and record the movement positions and morphological changes of the two types of cells in real time.

[0023] Since cells B will form cell spheroids after a period of growth and proliferation in the first cell culture chamber, the size of the cell spheroids is larger than the diameter of the medium output channel, so they will not flow out with the waste culture medium through the medium output channel.

[0024] Preferably, the monitored objects are the two types of cells and microtissues at the interface between the first cell culture chamber and the second cell culture chamber.

[0025] Preferably, in step (1), the microfluidic chip is incubated at 37°C for 24 h; in step (2), the microfluidic chip is cultured at 37°C for 24 h; in step (3), the microfluidic chip is placed with the first cell culture chamber on the top and the second cell culture chamber on the bottom, the cell culture chambers are subjected to real-time medium exchange, continuous culture is carried out at 37°C for 72 h, and photographs are taken every 30 min using the CO2 microscope stage of the live cell workstation.

[0026] Preferably, the monitored objects are the two types of cells and microtissues at the interface between the first cell culture chamber and the second cell culture chamber.

[0027] The cover glass of the microfluidic chip provided by the present invention is used for sealing and fluorescence imaging observation; the bottom plate glass is common optical glass, which is used for sealing and forming a flow channel and chamber system together with the PDMS membrane. The cell culture chamber part of the PDMS membrane is in a shape of two connected squares of different sizes arranged vertically, and is divided into two cell culture chambers by a pluggable movable partition, which can assist the gel culture medium in the second cell culture chamber to form a horizontal interface during the solidification process.

[0028] The solid culture gel in the microfluidic chip provided by the present invention is a hydrogel formed by mixing a polymer and a crosslinking agent, the polymer is polyvinyl alcohol or dextran, the crosslinking agent is thiol polyethylene glycol or cyclodextrin, which is a viscous transparent liquid at normal temperature. When the crosslinking agent is added and the temperature reaches 37°C, incubation for 30 min will trigger chemical crosslinking to form a solid transparent gel, which supports cell imaging analysis; the liquid culture gel is polyvinyl alcohol or dextran, excluding the crosslinking agent.

[0029] Solid gels can simulate the epithelial tissue layer and mucosal layer of organs in the body, while liquid gels can simulate interstitial fluid, serving as a medium for substance exchange between blood and tissue cells. Most interstitial fluid is in a gel state and cannot flow freely. Therefore, using a solid-liquid gel microfluidic chip can fully simulate the real state of in vivo tissues, which is beneficial for studying the interaction between free cells in the interstitial fluid and mucosal cells at the solid-liquid interface. In this invention, the first cell culture chamber is a liquid gel with a fast flow rate; a single culture medium inlet channel is sufficient to meet the cell's nutritional needs. The second culture chamber is a solid gel with poor flowability; multiple culture medium inlet channels are designed to facilitate sufficient substance exchange between the culture medium and the cells in the gel. Furthermore, all culture medium inlet channels are designed at the bottom to prevent disturbance to the cell state at the solid-liquid gel interface.

[0030] The beneficial effects of this invention are:

[0031] The microfluidic chip provided by this invention uses PDMS material for the culture layer, which has good biocompatibility. The top layer is made of quartz glass, which has no background fluorescence and excellent light transmittance. The cell culture chamber of the microfluidic chip is designed as a two-chamber structure with a clear solid-liquid gel interface area, allowing for vertical recording and observation of cell migration behavior. The microfluidic chip provided by this invention is fully functional, easy to operate, and can independently perform various fluorescence signal detections on the chip.

[0032] This invention utilizes microfluidic technology, employs cell immunofluorescence staining combined with confocal microscopy to image and monitor migrating cells, enabling behavioral analysis of cell spheres of different sizes during movement and observation of morphological changes within the cell spheres. Attached Figure Description

[0033] Figure 1 : A schematic diagram of the microfluidic chip structure of the present invention;

[0034] Figure 2 : A schematic diagram of the PDMS film structure of the microfluidic chip of the present invention;

[0035] Figure 3 The steps for studying cell interaction processes using the microfluidic chip of the present invention;

[0036] Figure 4 The morphology of two types of cells at the gel interface under three-dimensional culture conditions using the microfluidic chip of the present invention.

[0037] The diagram shows: 1. Cover glass, 2. PDMS membrane, 3. Base glass, 4. First cell culture chamber, 5. Second cell culture chamber, 6. Partition, 7. First fresh culture medium tank, 8. Culture medium inlet channel, 9. Culture medium outlet channel, 10. First waste culture medium tank, 11. Second fresh culture medium tank, 12. Second waste culture medium tank, 13. Total culture medium inlet channel, 14. Sub-culture medium inlet channel. Detailed Implementation

[0038] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0039] As described in the background section, existing microfluidic chips are inconvenient to operate, have poor light transmittance, and produce unclear images during observation. Therefore, this invention provides a microfluidic chip for real-time monitoring of cell interaction processes, comprising, from top to bottom, a rectangular cover glass 1, a PDMS membrane 2, and a bottom glass 3. A cell culture chamber is located at the center of the PDMS membrane 2, and the cover glass 1 is positioned directly above the cell culture chamber. The area of ​​the cell culture chamber occupies 1 / 4 to 1 / 2 of the PDMS membrane area. The cell culture chamber is divided into rectangular, identically sized first microcells by a movable partition 6. The first cell culture chamber 4 comprises a cell culture chamber and a second cell culture chamber 5. The left side of the first cell culture chamber 4 is connected to the first fresh culture medium tank 7 via a culture medium input channel 8, and the right side of the first cell culture chamber 4 is connected to the first waste culture medium tank 10 via a culture medium output channel 9. The opposite sides of the second cell culture chamber 5 are respectively connected to the second waste culture medium tank 12 via culture medium output channels 9. The lower side of the second cell culture chamber 5 is connected to a total culture medium input channel 13 via several sub-culture medium input channels 14. Both ends of the total culture medium input channel 13 are respectively connected to the second fresh culture medium tank 11. The first fresh culture medium tank 7, the second fresh culture medium tank 11, and the second waste culture medium tank 12 are all aligned in a straight line.

[0040] The thickness of the cover glass 1 and the bottom glass 3 is 0.5-1.5mm, the thickness of the PDMS film 2 is 1.5-2.5mm, the PDMS film 2 and the bottom glass 3 are the same size, the width of the cover glass 1 is less than or equal to the width of the PDMS film 2, and the length of the cover glass 1 is less than the distance between the second waste culture medium pools 10.

[0041] Real-time monitoring of cell interaction processes using the aforementioned microfluidic chip includes the following steps:

[0042] (1) Solid culture gel and perfusion of cell A

[0043] The sterilized PDMS membrane 2 is pressed firmly against the base glass 3. A 1 mg / ml gel A working solution is prepared, and 10 μl of cell A suspension is added to the gel working solution and mixed thoroughly. Approximately 100 μl of the above mixture is then added to the second cell culture chamber 5 using a pipette until it is full. The microfluidic chip is placed in an incubator and incubated for 30 min for gel solidification. After gel solidification, the partition (6) is removed, and the cover glass 1 is placed above the cell culture chamber and pressed firmly. The microfluidic chip is then placed flat in an incubator at 37°C and incubated for 24 h.

[0044] (2) Liquid culture gel and perfusion of cells B

[0045] Prepare a 1 mg / ml liquid gel B working solution. Add 10 μl of cell B suspension to the gel B working solution and mix thoroughly. Use a syringe pump to inject the mixture into the fresh culture medium tank 7, which then flows through the culture medium inlet channel 8 into the first cell culture chamber 4 and fills the chamber. Place the perfused microfluidic chip horizontally in an incubator and incubate at 37°C for 24 h.

[0046] (3) Real-time monitoring of cell-chip interactions

[0047] Prepare fresh culture medium A and culture medium B. Connect the first fresh culture medium tank 7, the second fresh culture medium tank 11, the first waste culture medium tank 10, and the second waste culture medium tank 12 to Tygon@ tubing respectively. Use an injection pump to inject fresh culture medium A into the second cell culture chamber 5 through the second fresh culture medium tank 11, and inject fresh culture medium B into the first cell culture chamber 4 through the first fresh culture medium tank 7. The injection rate of cell culture chambers 4 and 5 is 250 μl / h. Waste culture medium flows out and is collected through the first waste culture medium tank 10 and the second waste culture medium tank 12.

[0048] The microfluidic chip was placed vertically in the live cell workstation incubator with the first cell culture chamber 4 on top and the second cell culture chamber 5 on the bottom. The cell culture chambers were continuously incubated at 37°C for 72 hours using an infusion pump. Long-term cell observation was performed using a CO2 microscopic stage of the live cell workstation, with images taken every 30 minutes. The interface between the first cell culture chamber 4 and the second cell culture chamber 5 was selected as the monitoring site to record the movement, position, and morphological changes of the two types of cells and micro-tissues in real time.

[0049] To enable those skilled in the art to better understand the technical solution of this application, the technical solution of this application will be described in detail below with reference to specific embodiments.

[0050] The test materials used in the embodiments of this invention are all conventional test materials in the art and can be purchased through commercial channels.

[0051] Example

[0052] The microfluidic chip prepared by this invention is as follows: Figure 1 , Figure 2 As shown, the structure includes a rectangular cover glass 1, a PDMS membrane 2, and a bottom glass 3 arranged sequentially from top to bottom. A cell culture chamber is located at the center of the PDMS membrane 2, and the cover glass 1 is positioned directly above the cell culture chamber. The cell culture chamber occupies 1 / 4 of the area of ​​the PDMS membrane. The cell culture chamber is divided into a first cell culture chamber 4 and a second cell culture chamber 5 of the same rectangular size by a movable partition 6. The left side of the first cell culture chamber 4 is connected to a first fresh culture medium pool 7 via a culture medium input channel 8, and the right side of the first cell culture chamber 4 is connected to a first waste culture medium pool 10 via a culture medium output channel 9. The opposite sides of the second cell culture chamber 5 are respectively connected to a second waste culture medium pool 12 via culture medium output channels 9. The lower side of the second cell culture chamber 5 is connected to a total culture medium input channel 13 via several sub-culture medium input channels 14. The two ends of the total culture medium input channel 13 are respectively connected to a second fresh culture medium pool 11. The first fresh culture medium tank 7, the second fresh culture medium tank 11, and the second waste culture medium tank 12 are aligned in a straight line. The second fresh culture medium tank 11, the first waste culture medium tank 10, and the second waste culture medium tank 12 are also aligned in a straight line. The cover glass 1 and the base glass 3 are 1 mm thick, the PDMS film 2 is 2 mm thick, the PDMS film 2 and the base glass 3 are the same size, the width of the cover glass 1 is less than or equal to the width of the PDMS film 2, and the length of the cover glass 1 is less than the distance between the second waste culture medium tanks 10.

[0053] Real-time monitoring of cell interaction processes using the microfluidic chip prepared according to the present invention includes the following steps:

[0054] (1) Solid culture gel and perfusion of HEK 293T cells

[0055] The sterilized PDMS membrane 2 was pressed firmly against the glass substrate 3. A working solution of gel A (50 mmol / L polyvinyl alcohol and thiocyclodextrin) was prepared using 0.5 mL of culture medium A (DMEM high-glucose medium + 10% fetal bovine serum + 1% antibiotics). Then, 0.5 mL of 293T cell suspension was added and thoroughly mixed. 100 μL of this mixture was pipetted into the second cell culture chamber 5 until it was full. The microfluidic chip was placed in an incubator and incubated for 30 min to allow gel solidification, resulting in a transparent, elastomer gel. The septum 6 was removed to avoid damaging the gel, which could then self-support and fill the lower cell culture chamber. The cover glass was then attached. The microfluidic chip was placed flat in an incubator at 37°C and incubated for 24 h.

[0056] (2) Perfusion of liquid culture gel and mesenchymal stem cells

[0057] Prepare a 30 mmol / L (polyvinyl alcohol) working solution for liquid gel B using 0.5 mL of culture medium B (a-MEM medium + 10% fetal bovine serum + 1% penicillin-dextrose antibody). Then add 0.5 mL of mesenchymal stem cell suspension and mix thoroughly. Connect the fresh culture medium pool 7 on the left side of the first cell culture chamber 4 to a 1.5 mm diameter Tygon@ tubing. Use a syringe pump to inject liquid gel B into the first cell culture chamber 4 until it is full. Place the perfused microfluidic chip horizontally in an incubator and incubate at 37°C for 24 h. A clear solid-liquid interface layer is formed between liquid gel B and the solidified gel A.

[0058] (3) Real-time monitoring of cell-chip interactions

[0059] Connect the second fresh culture medium pools 11 on the left and right sides of the bottom of the second cell culture chamber 5 to Tygon@ tubing with a diameter of 1.5 mm. Use an injection pump to inject fresh culture medium A into the second cell culture chamber 5 through the bottom multi-component culture medium inlet channel 14. Waste liquid is collected through the culture medium outlet channels 9 on the left and right sides of the second cell culture chamber 5 and the second waste culture medium pool 12. The injection rate is 250 μl / h, and the culture medium turnover rate in the culture chamber is maintained at 6.0 mL / d.

[0060] Connect the first fresh culture medium pool 7 on the left side of the first cell culture chamber 4 to a Tygon@ tubing with a diameter of 1.5 mm. Use a syringe pump to inject fresh culture medium B through the culture medium inlet channel 8 on the left side of the first cell culture chamber 4. Waste liquid is collected through the culture medium outlet channel 9 on the right side and the waste culture medium pool 10. The injection rate is 250 μl / h, and the culture medium turnover rate in the culture chamber is maintained at 6.0 mL / d.

[0061] The microfluidic chip was moved into the live cell workstation incubator, placed vertically and fixed with the first cell culture chamber 4 above and the second cell culture chamber 5 below. The cell culture chambers were continuously circulated using an infusion pump. The instrument was turned on, and air and carbon dioxide cylinders were simultaneously opened. The valves were adjusted to maintain a CO2 concentration of 5% in the incubator. The temperature was adjusted to 37℃ for continuous incubation for 72 hours. The microscope was turned on, and the focus and imaging parameters were adjusted. The interface between the first cell culture chamber 4 and the second cell culture chamber 5 was selected as the monitoring site, and images were taken every 30 minutes. After 72 hours, as shown... Figure 4 As shown, cell morphology changes with increasing culture time; 293T cells appear as round epithelial cells, while mesenchymal stem cells are spindle-shaped, and the cells exhibit characteristics of a three-dimensional culture state.

[0062] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A method for real-time monitoring of cell interaction processes using a microfluidic chip for real-time monitoring of cell interaction processes, characterized in that, Includes the following steps: (1) Solid culture gel and perfusion of cell A The PDMS membrane was attached to the base glass. The working solution of gel A and the suspension of cell A were mixed evenly and injected into the second cell culture chamber until it was full. Gel A was cured. After curing, the partition was removed and the cover glass was placed above the cell culture chamber to culture the microfluidic chip. (2) Liquid culture gel and perfusion of cells B After mixing gel B working solution and cell B suspension evenly, inject the mixture into the first cell culture chamber until it is full, and then culture the microfluidic chip. (3) Real-time monitoring of cell-chip interactions Culture medium A is injected into the second cell culture chamber through the second fresh culture medium pool, and culture medium B is injected into the first cell culture chamber through the first fresh culture medium pool. Waste culture medium is collected by flowing out through the waste culture medium pool. The microfluidic chip is placed vertically in the live cell workstation to monitor the interface between the first and second cell culture chambers and record the movement position and morphological changes of the two types of cells in real time. The microfluidic chip for real-time monitoring of cell interaction processes includes a PDMS membrane, with a cell culture chamber at the center of the PDMS membrane. The cell culture chamber is divided into a first cell culture chamber and a second cell culture chamber. One side of the first cell culture chamber is connected to a first fresh culture medium tank, and the opposite side of the first cell culture chamber is connected to a first waste culture medium tank. The opposite sides of the second cell culture chamber are respectively connected to a second waste culture medium tank, and the bottom of the second cell culture chamber is connected to a second fresh culture medium tank. The cell culture chamber is topped with a cover glass plate and the PDMS membrane is bottomed with a base glass plate. The area of ​​the cell culture chamber is 1 / 4 to 1 / 2 of the area of ​​the PDMS membrane. The cell culture chamber is divided into a first cell culture chamber and a second cell culture chamber by a movable partition. The first cell culture chamber and the second cell culture chamber are rectangular and the same size.

2. The method as described in claim 1, characterized in that, The bottom of the second cell culture chamber is connected to the second fresh culture medium pool via a culture medium input channel. The culture medium input channel is divided into a total culture medium input channel and several sub-culture medium input channels. The sub-culture medium input channels are connected to the bottom of the second cell culture chamber, and the two ends of the total culture medium input channel are connected to the second fresh culture medium pool respectively.

3. The method as described in claim 1, characterized in that, The first fresh culture medium pool, the second fresh culture medium pool, and the second waste culture medium pool are in a straight line.

4. The method as described in claim 1, characterized in that, The cover glass is made of quartz glass. The cover glass, PDMS film, and base glass are all rectangular. The thickness of the cover glass and base glass is 0.5-1.5 mm, the thickness of the PDMS film is 1.5-2.5 mm, the PDMS film and base glass are the same size, the width of the cover glass is less than or equal to the width of the PDMS film, and the length of the cover glass is less than the distance between the second waste culture medium tanks.

5. The method as described in claim 1, characterized in that, The monitored objects are two types of cells and micro-tissues at the interface between the first and second cell culture chambers.

6. The method as described in claim 1, characterized in that, In step (1), the microfluidic chip is cultured at 37°C for 24 hours; in step (2), the microfluidic chip is cultured at 37°C for 24 hours; in step (3), the microfluidic chip is placed with the first cell culture chamber on top and the second cell culture chamber on the bottom, the cell culture chamber is changed in real time, and the microfluidic chip is continuously cultured at 37°C for 72 hours. The microscopy stage of the live cell workstation is used to take pictures every 30 minutes.

Images