Microfluidic cell chip and virus isolation culture method based on the same

Abstract

The application discloses a micro-fluidic cell chip and a virus separation and culture method based on the cell chip. The micro-fluidic cell chip comprises a metal frame, a lower cover plate, a chip frame, a core chip and an upper cover plate, the core chip is provided with a plurality of cell culture cavities, a main flow channel is arranged on the surface of the core chip, and the main flow channel is communicated with the plurality of cell culture cavities. The virus separation and culture method based on the micro-fluidic cell chip adopts a plurality of cell lines for co-culture, realizes the sequential incubation of one sample to be analyzed and different cell lines, can significantly save clinical samples, maximally improves sample utilization, simultaneously reduces the consumption of culture reagents, shortens the separation and identification time, and theoretically realizes the culture of at least ten different cell lines at the same time due to the design of the micro-fluidic cell chip provided by the application, significantly increases the throughput of virus sensitive host cell screening, and realizes the high-throughput, automatic and rapid screening of virus sensitive cell lines.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological science, and particularly relates to a microfluidic cell chip and a virus isolation culture method based on the cell chip. BACKGROUND

[0002] At present, new and recurrent viral infectious diseases still pose a serious threat to human life and property. Therefore, rapid identification of viruses is crucial for early diagnosis of diseases and epidemic prevention and control. It is well known that the virus plaque assay based on live cell culture is the gold standard for virus diagnosis and follows the "Koch's rule". When a new viral infectious disease occurs, an important problem is to identify the permissive host cell line of the virus through the plaque assay, which is particularly crucial for subsequent virus amplification, virus isolation culture and vaccine production.

[0003] The existing conventional virus isolation culture method is usually carried out in a polystyrene well plate or a cell culture flask, and the specific operation includes specific cell line culture, cell inoculation, virus infection, lesion observation and virus harvesting. The number of cells required by this traditional culture method is usually large (10 5 -10 8 ), the amount of reagents such as culture medium is large, the experimental period is long (7-10 days), and the sensitivity is low; for major viral infectious diseases, the clinical samples are often precious, and when the viral load in the sample is low, the possibility of failure of isolation culture is great; if the isolated virus lacks an in vitro culture system (such as human norovirus), it is impossible to develop effective antiviral strategies. At present, when isolating and identifying viruses through cell culture and infection, different cell lines are inoculated and then infected separately in independent cell culture flasks or in the same microplate, which is tedious and prone to cross contamination, and cannot realize rapid and high-throughput screening of virus permissive host cells and rapid isolation and culture of viruses.

[0004] In actual work, it is difficult to obtain samples infected by major viral infectious diseases, and even if the virus-containing samples are obtained, the viral load is very low after processing, and it is almost impossible to isolate and culture them by conventional methods, which greatly hinders the subsequent classification, identification and vaccine development of viruses, and wastes precious virus samples.

[0005] Therefore, the present application is proposed. SUMMARY

[0006] The microfluidic cell chip focuses on developing a micro device and a technical method which can realize controllable and accurate cell culture and biochemical analysis on a micro scale, and multiple cell lines can be co-cultured in the same microfluidic cell chip, so that the consumption of reagents can be reduced, the risk of pollution can be reduced, and the sample throughput processing capacity can be increased.

[0007] In order to achieve the above-mentioned purpose, the microfluidic cell chip for virus isolation culture provided by the present application comprises a metal frame, a lower cover plate, a chip frame, a core chip and an upper cover plate in the order of bottom-up assembly, the chip frame is used for fixing the position of the core chip, the lower cover plate is used for supporting the bottom surface of the core chip, the core chip is provided with a plurality of cell culture cavities, and the upper cover plate is used for sealing the cell culture cavities.

[0008] Further, the metal frame, the lower cover plate, the chip frame, the core chip and the upper cover plate are fixedly connected through screws.

[0009] Preferably, the main flow channel is U-shaped, and the plurality of cell culture cavities are arranged in two parallel rows.

[0010] Preferably, the top end of the main flow channel is kept at the same height as the top end of the cell culture cavities.

[0011] Preferably, the upper cover plate and the lower cover plate are made of polymethyl methacrylate, the core chip is a polydimethylsiloxane chip, and the chip frame is a polylactic acid frame.

[0012] The present application also provides a virus isolation culture method based on the microfluidic cell chip, which is operated on the microfluidic cell chip for virus isolation culture.

[0013] (1) coating the core chip with polylysine with a concentration of 0.1 mg / mL, incubating overnight at 4 DEG C and then washing;

[0014] (2) inoculating cells in the cell culture cavities and injecting cell culture medium for culture until the cells are completely adhered;

[0015] (3) discharging the cell culture medium, washing the cells with phosphate buffered saline solution, and discharging the phosphate buffered saline solution;

[0016] (4) the virus dilution liquid is injected into the cell culture chamber, incubated at 37℃ with 5% CO2 for 2h, then the infected virus dilution liquid is discharged for recovery and re-injected into the cell culture chamber for continuous perfusion infection, the virus dilution liquid is recovered and re-injected into the microfluidic cell chip for infection, and the cycle is repeated for 3 times.

[0017] Further, in step (2), the cell culture medium is injected into the cell culture chamber through the liquid inlet and discharged from the liquid outlet after perfusion of the cell culture chamber, wherein the cell culture medium is continuously perfused at a flow rate of 100 μL / h to update the cell culture medium.

[0018] Further, the cell culture medium and the virus dilution liquid are injected into the cell culture chamber by a peristaltic pump.

[0019] Preferably, in step (4), the infected virus dilution liquid is discharged for recovery and re-injected into the cell culture chamber at a flow rate of 2 mL / h for continuous perfusion infection.

[0020] The microfluidic cell chip and the virus isolation and culture method based on the cell chip provided by the application have the following beneficial effects:

[0021] 1. The microfluidic cell chip can reduce material consumption, optimize interaction, shorten sample processing time, and reduce cost. The core microfluidic cell chip provided by the application can be repeatedly operated and used, which can reduce environmental pollution while reducing production cost. The microfluidic cell chip focuses on the development of micro devices and technical methods that can realize controllable and accurate cell culture and biochemical analysis on a micro scale. Compared with traditional cell culture methods, the co-culture of multiple cell lines in the application can be realized in the same microfluidic cell chip, and the consumption of reagents can be reduced, the risk of pollution can be reduced, and the throughput of sample processing can be increased. By continuous perfusion culture or establishing a chemical gradient, the natural microenvironment of cells can be more realistically simulated. The microfluidic cell chip for virus isolation and culture can miniaturize, integrate, automate, and parallelize conventional biochemical processes, and by directly coupling downstream analytical chemistry instruments, a small amount of cells or even a single cell can be studied with high temporal / spatial resolution.

[0022] 2. Even if the amount of virus sample is very limited, the utilization rate of the sample can be improved by the cyclic perfusion infection method of the application, and a virus isolation and culture scheme can be established using a small amount of sample to maximize the rapid screening of sensitive host cell lines of viruses.

[0023] 3. The virus isolation and culture method based on the microfluidic cell chip, which adopts the repeated cycle infection method, can significantly save the clinical sample, maximize the sample utilization rate, reduce the consumption of culture reagents, shorten the isolation and identification time, and significantly increase the throughput of virus-sensitive host cell screening. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 FIG. 1 is a structural schematic diagram of the microfluidic cell chip in the embodiment.

[0025] Figure 2 FIG. 2 is a front view of the microfluidic cell chip in the embodiment.

[0026] Figure 3 FIG. 3 is a top view of the microfluidic cell chip in the embodiment.

[0027] Figure 4 FIG. 4 is a bright field inverted microscope photo of the EV71-sensitive host cell in Example 1 after simulated screening in a 96-well plate (MOI = 40.0).

[0028] Figure 5 FIG. 5 is a bright field inverted microscope photo of the EV71-sensitive host cell in Example 1 after simulated screening in the microfluidic cell chip (MOI = 0.389).

[0029] Figure 6 FIG. 6 is a bright field inverted microscope photo of the EV71-sensitive host cell in Example 2 after simulated screening in the microfluidic cell chip (MOI = 3.89 x 10 -4 ).

[0030] Figure 7 FIG. 7 is a bright field inverted microscope photo of the H1N1-sensitive host cell in Example 3 after simulated screening in a 96-well plate (MOI = 12.3).

[0031] Figure 8 FIG. 8 is a bright field inverted microscope photo of the H1N1-sensitive host cell in Example 3 after simulated screening in the microfluidic cell chip (MOI = 12.3).

[0032] Figure 9 FIG. 9 is a viral nucleic acid fluorescence quantitative PCR amplification curve of the H1N1-sensitive host cell in Example 4 after infection (MOI = 0.012).

[0033] Figure 10 The graph shows the quantitative real-time PCR amplification curve of viral nucleic acid after H1N1-sensitive host cells were infected in Example 5 (MOI = 0.00012).

[0034] In the picture:

[0035] 1. Metal frame, 2. Lower cover plate, 3. Chip frame, 4. Core chip, 5. Upper cover plate, 6. Screws, 7. Cell culture chamber, 8. Main channel, 9. Liquid inlet, 10. Liquid outlet. Detailed Implementation

[0036] To enable those skilled in the art to better understand the present invention, the present invention will be further described in detail below with reference to specific embodiments.

[0037] like Figures 1-3 As shown, a microfluidic cell chip for virus isolation and culture comprises five assembly components in a bottom-up assembly order: a metal frame 1, a lower cover plate 2, a chip frame 3, a core chip 4, and an upper cover plate 5. These five components are fixedly connected by screws 6. It can be used for the co-culture of at least ten different cell lines, enabling high-throughput screening and identification of virus-sensitive host cell lines while significantly reducing sample volume.

[0038] The core chip 4 measures 89.0mm × 49.0mm × 12.0mm (length × width × height). Inside the core chip 4 are multiple cylindrical cell culture chambers 7, each measuring 6.0mm × 2.0mm (diameter × depth). The core chip 4 is designed as a unidirectional flow channel, with a recessed main flow channel 8 on its surface, without branching channels. The main flow channel 8 is U-shaped and meandering, used for the flow of cell culture medium and virus diluent. At each end of the main flow channel 8 are an inlet 9 and an outlet 10, serving as the culture medium supply unit and waste liquid outlet, respectively. Both the inlet 9 and outlet 10 measure 3.5mm × 0.9mm (diameter × depth), and their functions are interchangeable.

[0039] The main channel 8 measures 0.9mm × 0.9mm (width × depth), and its top end is at the same height as the cell culture chamber 7. The main channel 8 can connect multiple cell culture chambers 7, for example, 10 or more. The cell culture chambers 7 are arranged in two parallel rows, with 5 or more cell culture chambers 7 distributed in each row. The spacing between each cell culture chamber 7 is 4.0mm, and the distance between each row of cell culture chambers 7 is 5.5mm.

[0040] The upper cover plate 5 and the lower cover plate 2 are made of polymethyl methacrylate (PMMA) and are made by laser cutting process, high light transmittance, not easy to deformation. The upper cover plate 5 is used to close the cell culture chamber 7, and the upper cover plate completely closes the main flow channel and the cell culture chamber, and the lower cover plate 2 is used to support the bottom surface of the core chip 4. The core chip 4 is a microfluidic cell culture chip (PDMS chip) constructed by designing and processing polydimethylsiloxane (polydimethylsiloxane, [C2H6OSi] n , PDMS) and is used for virus-permissive host cell screening and virus microculture. The PDMS chip is prepared by 3D printing injection molding in a reverse molding manner, and the co-culture of multiple different cell lines is realized in the PDMS chip. The PDMS chip has the characteristics of high transparency, elasticity, air permeability, good chemical inertness, strong repeatability and the like. The chip frame 3 is made of polylactic acid (PLA) frame, which is made by 3D printing process and is clamped between the upper cover plate 5 and the lower cover plate 2, and is used to fix the center position of the PDMS chip. The metal frame 1 at the bottom is designed and processed by computer numerical control technology, so that the entire microfluidic cell chip has strong rigidity, and the structural integrity of the entire virus separation and culture microfluidic cell chip is ensured.

[0041] The microfluidic cell chip for virus separation and culture can reduce material consumption, optimize interaction, shorten sample processing time and reduce cost. The microfluidic cell chip focuses on developing micro devices and technical methods that can realize controllable and accurate cell culture and biochemical analysis on a micro scale. Compared with traditional cell culture methods, the co-culture of multiple cell lines in the present application can be realized in the same microfluidic cell chip, and the consumption of reagents can be reduced, the risk of pollution can be reduced, and the throughput of sample processing can be increased. By continuous perfusion culture or establishing a chemical gradient, the natural microenvironment of cells can be more truly simulated. The microfluidic cell chip for virus separation and culture can miniaturize, integrate, automate and parallelize the conventional biochemical process, and by directly coupling downstream analytical chemistry instruments, a small amount of cells or even a single cell can be studied with high time / space resolution.

[0042] A virus separation and culture method based on a microfluidic cell chip, comprising the following steps:

[0043] 1. Coating and culturing cells in the microfluidic cell chip for virus separation and culture, and completely adhering.

[0044] The microfluidic cell chip for virus separation and culture is a one-way fluid chip. Before inoculating cells, the microfluidic cell chip needs to be sterilized by high-pressure steam, and after sterilization, it is dried, and the core chip 4 is coated with polylysine (working concentration of 0.1 mg / mL), and after 4℃ overnight incubation, it is washed with sterile ultrapure water. The number of cells contained in the cell culture chamber 7 is 1.0 x 104 ~4.0 x 10 4 The cells can be cultured to full confluence after inoculation. The cell culture medium is driven by positive pressure peristaltic pump and injected from the inlet 9, and discharged from the outlet 10 after perfusing the cell culture chamber 7. The cells are continuously perfused with a flow rate of 100 μL / h to update the medium and ensure sufficient nutrients and oxygen when the cells grow in the microfluidic cell chip.

[0045] 2. Virus culture and microscale amplification.

[0046] The virus culture and microscale amplification can be performed after the cells are fully confluent in the microfluidic cell chip. The cell culture medium in the microfluidic cell chip is discharged by a peristaltic pump at a flow rate of 2 mL / h, and the cells are washed with phosphate buffered saline (PBS) at the same flow rate. After the residual PBS in the chip is discharged, the virus dilution solution with a specific multiplicity of infection (MOI) is pumped into the cell culture chamber 7, and incubated at 37°C with a volume fraction of 5% CO2 for 2 h. The multiplicity of infection can be high, low, or very low. Then the infected virus dilution solution in the microfluidic cell chip is discharged and recovered, and the recovered virus dilution solution is continuously pumped into the cell culture chamber 7 by a peristaltic pump at a flow rate of 2 mL / h for continuous perfusion infection. The virus dilution solution is recovered multiple times, and continuously perfused according to the above scheme. The virus dilution solution is recovered and infected with cells in the microfluidic cell chip again, and the cycle is repeated for 3 times. Even if the amount of virus sample is very limited, the utilization rate of the sample can be improved by the cyclic perfusion infection method of the present application, and a microscale sample can be used to establish a virus isolation culture scheme, and the sensitive host cell line of the virus can be quickly screened to the maximum extent.

[0047] The microfluidic cell chip of the present application can culture multiple cell lines at the same time, which can greatly increase the infection probability of unknown samples and screen the corresponding host cell line.

[0048] 3. Observe the virus-infected cells and take photos.

[0049] After a specific time point of infection, such as 72 h, the perfusion culture is stopped. First, the cytopathic effect is observed with an inverted microscope and photographed, and the sensitive cell line is preliminarily judged. Then the fixing screw 6 of the microfluidic cell chip is carefully disassembled, and the upper cover plate 5 is lifted to expose the PDMS chip. The cells in the cell culture chamber 7 are scraped with a cell scraper, and the residual medium and cells are collected to detect the number of virus nucleic acid copies to confirm successful infection.

[0050] The virus isolation and culture method based on the microfluidic cell chip provided in the application can significantly save clinical samples and maximize the utilization of samples by using the repeated cycle infection method, and can reduce the consumption of culture reagents and shorten the isolation and identification time. Since the microfluidic cell chip provided in the application is designed to contain multiple built-in cell culture chambers 7, theoretically, at least ten different cell lines can be cultured at the same time, which significantly increases the throughput of virus-sensitive host cell screening. The screening of virus-sensitive cell lines is performed in a closed chip system, which is not easy to be contaminated.

[0051] The technical method provided in the application can replace the traditional virus isolation and culture and cell culture methods, and can realize high-throughput and rapid screening of virus-sensitive cell lines. In addition, the core microfluidic cell chip provided in the application can be repeatedly used, which can reduce the production cost and reduce environmental pollution.

[0052] Example 1, simulation screening of enterovirus EV71 (Chinese vaccine strain, GenBank No. HQ328793) in a microfluidic cell chip at a low multiplicity of infection (MOI).

[0053] The cell line can be a conventional virus amplification cell line, or an in vitro culture cell line derived from other species.

[0054] (1) EV71 typical CPE (cytopathic effect) characterization: human lung cancer cells (A549), African green monkey kidney cells (Vero cells) and human malignant embryonal rhabdomyosarcoma cells (RD cells) were inoculated at 1×10 4 The cells were inoculated into a 96-well plate at a cell number of 1×10 Figure 4

[0055] (2) Low MOI infection: A549, Vero and RD cells were inoculated at 1×10 4 The cells were inoculated into the cell culture chamber 7 of the microfluidic cell chip at a cell number of 1×10 Figure 5

[0056] Example 2, simulation screening of EV71 in a microfluidic cell chip at a very low MOI. ​​

[0057] The cell line can be a conventional virus amplification cell line, and can also be an in vitro cultured cell line from other species.

[0058] Low MOI infection: A549, Vero and RD cells, 1 x 10 4 Cells were seeded into the microfluidic cell chip culture chamber at 37°C, 5% CO2 incubator for 12 h, and EV71 was used to infect at MOI = 3.89 x 10 -4 Infection was performed and cytopathic effect was observed under bright field inverted microscope at 48 h post-infection. The results showed that RD cells produced cytopathic effect earliest and most significantly, which was consistent with the results in 96-well plate in Example 1 (as shown in Figure 6

[0059] Example 3, High MOI screening of sensitive host cells for influenza virus (A / Puerto Rico / 8 / 1934, H1N1) in microfluidic cell chip.

[0060] The cell line can be a conventional virus amplification cell line, and can also be an in vitro cultured cell line from other species.

[0061] (1) H1N1 typical CPE characterization: A549, baby hamster kidney cells (BHK-21), Madin-Darby canine kidney cells (MDCK), Vero and RD cells, 1 x 10 4 Cells were seeded into 96-well plate at 37°C, 5% CO2 incubator for 12 h, and H1N1 was used to infect at MOI = 12.3; cytopathic effect was observed under bright field inverted microscope at 24 h post-infection. The results showed that MDCK cells produced obvious cytopathic effect, cell intrinsic morphology disappeared, cells dropped and produced vacuoles, and other cell lines had no obvious cytopathic effect (as shown in Figure 7

[0062] (2) High MOI infection: A549, BHK-21, MDCK, Vero and RD cells, 1 x 10 4 Cells were seeded into the microfluidic cell chip culture chamber 7 at 37°C, 5% CO2 incubator for 12 h, and H1N1 was used to infect at MOI = 12.3; cytopathic effect was observed under bright field inverted microscope at 12 h and 24 h post-infection. The results showed that MDCK cells produced the most obvious cytopathic effect, indicating that MDCK cells were most sensitive to H1N1, which was consistent with the results in 96-well plate (as shown in Figure 8

[0063] Example 4, Low MOI screening of sensitive host cells for influenza virus (H1N1) in microfluidic cell chip.

[0064] ​​​The cell line can be a conventional virus amplification cell line, and can also be an in vitro cultured cell line from other species.

[0065] Low MOI infection: A549, MDCK, Vero and RD cells were infected with 1 x 10 4 The cells were inoculated into the cell culture chamber 7 of the microfluidic cell chip, and cultured at 37°C in a 5% CO2 incubator for 12 h. H1N1 was infected at an MOI of 0.012. The cell supernatant was collected 72 h after infection, and the viral nucleic acid copy number was detected by one-step real-time fluorescent PCR using an influenza A virus nucleic acid detection kit (DAJY-001-24T, Zhongshan University Daan Gene Co., Ltd.). Sample preparation and amplification system refer to the kit instructions. Sample amplification was performed using a Bio-Rad CFX Connect real-time quantitative PCR instrument, and amplification data analysis was performed using CFX Manager (Version 3.1). The results showed that when H1N1 was infected at an MOI of 0.00012, the virus replicated and amplified in both the 96-well plate and the microfluidic cell chip, and the titer of the progeny virus produced in the microfluidic chip was slightly higher (as shown in Figure 9

[0066] Example 5, simulation screening of sensitive host cells for influenza virus (H1N1) at very low MOI in a microfluidic cell chip:

[0067] The cell line can be a conventional virus amplification cell line, and can also be an in vitro cultured cell line from other species.

[0068] Low MOI infection: A549, MDCK, Vero and RD cells were infected with 1 x 10 4 The cells were inoculated into the cell culture chamber 7 of the microfluidic cell chip, and cultured at 37°C in a 5% CO2 incubator for 12 h. H1N1 was infected at an MOI of 0.00012. The cell supernatant was collected 72 h after infection, and the viral nucleic acid copy number was detected by one-step real-time fluorescent PCR using an influenza A virus nucleic acid detection kit (DAJY-001-24T, Zhongshan University Daan Gene Co., Ltd.). Sample preparation and amplification system refer to the kit instructions. Sample amplification was performed using a Bio-Rad CFX Connect real-time quantitative PCR instrument, and amplification data analysis was performed using CFX Manager (Version 3.1). The results showed that when H1N1 was infected at an MOI of 0.00012, the virus replicated and amplified in both the 96-well plate and the microfluidic cell chip, and the titer of the progeny virus produced in the microfluidic chip was slightly higher (as shown in Figure 10

[0069] ​​The application selects PDMS as the basic material of the microfluidic cell chip, assembles the cell chip according to a specific installation sequence, and realizes the simulation screening of two modes of virus sensitive host cells, virus micro-cultivation and co-cultivation of various cell lines. The virus isolation and cultivation method has the characteristics of high throughput, automation and rapidness, and can replace the traditional virus cultivation method.

[0070] The specific examples are applied in the detailed description of the inventive concept, and the above description of the embodiments is only used to help understand the core idea of the application. It should be pointed out that any obvious modification, equivalent replacement or other improvement made by those skilled in the art without departing from the inventive concept should be included in the protection scope of the application.

Claims

1. A virus isolation culture method based on a microfluidic cell chip, characterized by, Operating on a microfluidic cell chip for virus isolation culture, The microfluidic cell chip for virus isolation culture comprises, in a top-down assembly order, a metal frame, a lower cover plate, a chip frame, a core chip, and an upper cover plate, the chip frame is used to fix the position of the core chip, the lower cover plate is used to support the bottom surface of the core chip, the core chip is provided with a plurality of cell culture chambers, and the upper cover plate is used to seal the cell culture chambers; a recessed one-way main flow channel is arranged on the surface of the core chip and used for the flow of cell culture medium and virus diluent, the main flow channel is communicated with the plurality of cell culture chambers and has no branch flow channel; and the two ends of the main flow channel are respectively provided with a liquid inlet and a liquid outlet. The main flow channel is in a U shape, and the plurality of cell culture chambers are arranged in two parallel rows. The operation comprises the following steps: (1) coating the core chip with polylysine at a concentration of 0.1 mg / mL, incubating overnight at 4°C, and then washing; (2) inoculating cells in the cell culture chambers and injecting cell culture medium for culture until the cells are completely adherent; (3) discharging the cell culture medium, washing the cells with a phosphate buffered saline solution, and discharging the phosphate buffered saline solution; (4) injecting virus diluent into the cell culture chambers, incubating at 37°C under a 5% CO2 condition for 2 hours, then discharging the infected virus diluent, re-injecting the virus diluent into the cell culture chambers for continuous perfusion infection, repeatedly recovering the virus diluent, and continuously perfusing according to the above scheme; the virus diluent is recovered and re-infects the cells in the microfluidic cell chip for 3 cycles.

2. The microfluidic cell-chip based virus isolation culture method according to claim 1, wherein, In step (2), the cell culture medium is injected into the liquid inlet, perfuses the cell culture chambers, and is discharged from the liquid outlet, wherein the cell culture medium is continuously perfused at a flow rate of 100 μL / h to update the cell culture medium. 3.The microfluidic cell-chip based virus isolation culture method according to claim 1, wherein, The cell culture medium and the virus diluent are injected into the cell culture chambers by a peristaltic pump.

4. The microfluidic cell-chip based virus isolation culture method according to claim 1, wherein, In step (4), the infected virus diluent is discharged, re-injected into the cell culture chambers at a flow rate of 2 mL / h for continuous perfusion infection, and then fresh medium is continuously perfused at a flow rate of 100 μL / h.

5. The method of claim 1, wherein, The metal frame, the lower cover plate, the chip frame, the core chip, and the upper cover plate are fixedly connected by screws.

6. The method of claim 1, wherein, The top end of the main flow channel is at the same height as the top end of the cell culture chamber.

7. The method of claim 1, wherein, The upper cover plate and the lower cover plate are made of polymethyl methacrylate, the core chip is a polydimethylsiloxane chip, and the chip frame is a polylactic acid frame.

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