Ozone gas-liquid cell contamination method based on Transwell chamber

Abstract

The invention discloses an ozone gas-liquid cell contamination method based on a Transwell chamber, and belongs to the technical field of in-vitro gas contamination. The invention provides an O3 gas-liquid exposure mode based on a Transwell chamber by improving the contamination mode, and achieves the effects of simplifying the experimental method, reducing the experimental cost and improving the contamination efficiency. The gas-liquid exposed interface is constructed by utilizing the Transwell chamber, and cell inoculation, culture and sampling methods are simple and convenient; the Transwell chamber and the cell culture box with the O3 generation and real-time concentration monitoring functions are simple in principle, easy to understand and easy to obtain, and the O3 concentration can be monitored in real time; the experimental device is small in occupied space, low in building cost, good in operability and high in practicability.

Description

TECHNICAL FIELD

[0001] The application relates to an ozone gas-liquid exposure cell method based on a Transwell chamber and belongs to the technical field of in-vitro gas exposure. BACKGROUND

[0002] In recent years, atmospheric ozone (O3) pollution has become increasingly severe, mainly caused by photochemical reactions of nitrogen oxides and volatile organic compounds in motor vehicle exhaust and industrial emissions. O3 has strong oxidizing ability and can directly damage the respiratory tract and alveolar tissue after inhalation. Its harm mechanism mainly includes inducing excessive production of reactive oxygen species (ROS) leading to oxidative stress, inducing inflammatory response, damaging epithelial cell barrier integrity, damaging cell DNA and protein, etc. Long-term or repeated exposure to high-concentration O3 environment is closely related to significantly increased risk of various respiratory diseases, cardiovascular diseases and nervous system diseases, which poses a serious threat to public health.

[0003] In environmental toxicology research, animal experiments are costly, time-consuming and have uncertainties in extrapolating to humans due to species differences. Human exposure research has significant ethical limitations and implementation difficulties. Therefore, it is crucial to establish a reliable, controllable and highly physiological relevant in-vitro cell exposure model to further explore the health hazards and molecular mechanisms of O3. In-vitro cell experiments can directly observe the toxicological effects of O3 on different cell types of lung tissue and changes in their molecular mechanisms by precisely controlling exposure conditions such as exposure concentration and exposure time, which is an indispensable key model for screening potential toxic targets, finding prevention and treatment measures, evaluating prevention and treatment effects, and researching toxic effect mechanisms. Therefore, developing in-vitro exposure techniques that are closer to in-vivo physiological conditions, especially cell exposure models that can effectively simulate the microenvironment of the gas-liquid interface of the human respiratory system, has great significance for improving the scientific nature of O3 toxic effect research.

[0004] Due to the unstable nature of O3, which has strong oxidizing properties, there is no clear biomarker for O3 exposure. O3 exposure can cause the massive production of ROS and induce oxidative stress, among which ROS, superoxide dismutase (SOD) and glutathione (GSH) are key indicators of oxidative stress response. In addition, observation of cell morphological characteristics is one of the important means to evaluate the physiological state of the exposure model.

[0005] Currently, the main methods of gas cell exposure are as follows: (1) Immersion direct ventilation method: gas is directly introduced and dissolved in cell culture medium for exposure: such as introducing cigarette smoke and dissolving it in cell culture medium, diluting it for cell exposure, thereby exploring the role and molecular mechanism of respiratory system damage caused by cigarette smoke exposure in in vitro experiments; (2) Microfluidic biochip method: using microfluidic biochip and cell culture system to construct cell gas-liquid interface for gas exposure: such as administering drugs to biomimetic lung gas-liquid exposure, etc., to simulate the microenvironment of lung gas-liquid exchange in the in vitro environment; (3) Single concentration or multi-concentration cell gas-liquid interface exposure device: using this device to realize the in vitro cell single-pore or multi-pore exposure of inhalable substances.

[0006] However, due to the strong oxidizing properties and slight solubility of O3 in water, it is extremely unstable in the liquid phase and can rapidly decompose into reactive oxygen species (ROS). Therefore, in existing immersion direct ventilation exposure methods, the actual cell exposure dose is uncontrollable, and it is impossible to determine whether the cytotoxic effect after O3 exposure is directly caused by O3 or indirectly caused by intermediate products in the culture medium. Microfluidic biochips are complex to design and manufacture, costly, and inconvenient to operate such as cell seeding and sampling. Single-concentration or multi-concentration cell exposure devices require large spaces, are expensive, difficult to operate, and have high requirements for sterility in the experimental environment.

[0007] Therefore, developing a method that indirectly reflects the effects of O3 exposure based on changes in cell morphology characteristics and oxidative stress indicators has extremely high practical and economic value. Summary of the Invention

[0008] To address the aforementioned issues, this invention improves the exposure method by providing an O3 gas-liquid exposure method based on a Transwell chamber, thereby simplifying the experimental procedure, reducing experimental costs, and increasing exposure efficiency.

[0009] One object of the present invention is to provide a method for cell O3 gas-liquid exposure based on a Transwell chamber, comprising the steps of: (1) Seed cells in a Transwell chamber, add cell culture medium to the lower chamber, and incubate in a cell culture incubator; (2) After the cells adhere to the wall, remove the culture medium in the upper chamber, retain the culture medium in the lower chamber and contact the porous membrane of the Transwell chamber with the culture medium; place the Transwell chamber under O3 conditions.

[0010] In one embodiment, the number of cells seeded in step (1) is 1~3×10⁻⁶. 5 indivual.

[0011] In an embodiment, the medium added into the lower chamber in step (1) is a medium required for inoculating cells, such as Gibco RPMI 1640 medium or Gibco DMEM medium.

[0012] In an embodiment, the cells in step (1) include mouse alveolar epithelial cells MLE12, human bronchial epithelial cells Beas-2B, and mouse monocyte macrophages RAW264.7, etc.

[0013] In an embodiment, the O3 condition treatment in step (2) is a treatment for 1-5 h under an O3 concentration of 0.1-0.8 ppm.

[0014] In an embodiment, the O3 condition treatment in step (2) is a treatment for 1-5 h under an O3 concentration of 0.1-0.8 ppm.

[0015] In an embodiment, the O3 condition treatment in step (2) is a treatment for 1-5 h under an O3 concentration of 0.1-0.8 ppm.

[0016] In an embodiment, after the O3 condition treatment in step (2), the damage state of the cells under the O3 condition can be evaluated by observing the morphology of the cells or detecting the expression levels of ROS, GSH, and SOD in the cells.

[0017] The two objects of the present application are to provide the use of any of the above-described methods in screening biomarkers of O3 exposure.

[0018] The three objects of the present application are to provide the use of any of the above-described methods in screening drugs for alleviating damage caused by O3 exposure.

[0019] Advantages of the present application The present application improves the exposure method and provides an O3 gas-liquid exposure method based on a Transwell chamber, which simplifies the experimental method, reduces the experimental cost, and improves the exposure efficiency.

[0020] Specifically: (1) The Transwell chamber is used to construct a gas-liquid exposure interface, and the cell inoculation, culture, and sampling methods are simple; (2) The required Transwell chamber and cell incubator with O3 generation and real-time concentration monitoring are simple in principle, easy to understand, easy to obtain, and can realize real-time monitoring of O3 concentration; (3) The experimental equipment device occupies a small space, has low construction cost, is good in operability, and is highly practical. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1Schematic diagram of O3 gas-liquid exposure cell model; Figure 2 Effects of O3 gas-liquid exposure on MLE12 cell morphology; Figure 3 Effects of O3 gas-liquid exposure on MLE12 cell oxidative stress; Figure 4 Effects of different concentrations of O3 gas-liquid exposure on Beas-2B cell morphology; Figure 5 Effects of different concentrations of O3 gas-liquid exposure on Beas-2B cell oxidative stress.

[0022] Figure 6 Effects of different concentrations of O3 immersion direct ventilation exposure for different time on MLE12 cell morphology. DETAILED DESCRIPTION

[0023] The preferred embodiments of the present application are described below, and it should be understood that the embodiments are for better explaining the present application, and are not used to limit the present application.

[0024] In the case of the existing O3 cell exposure method, the present application considers the characteristics of O3, inoculates the cells to be exposed to the Transwell chamber, constructs the O3 gas-liquid interface, and exposes them to the O3 generation and real-time monitoring incubator. In this way, the O3 gas-liquid exposure is achieved, and compared with the traditional exposure method: (1) The top surface of the cell directly contacts O3, avoiding the mixed effects of intermediate products on the cell after contacting the cell culture medium; (2) The base surface of the cell contacts the cell culture medium through the Transwell porous membrane, effectively maintaining the normal physiological state and function of the cell, and synchronously simulating the physiological interface and microenvironment of the lung tissue in vivo; (3) The cell inoculation and sampling operation is simple, greatly improving the efficiency of in vitro O3 cell exposure.

[0025] Therefore, the gas-liquid exposure method based on the Transwell chamber is crucial for the exposure of gaseous substances.

[0026] The application utilizes a Transwell chamber with a diameter of 24 mm, a pore size of 0.4 μm, and a film material of polyethylene glycol terephthalate (PET) to construct a gas-liquid interface. The applied O3 exposure cell incubator (CE-Ozone, Shanghai Ta Wang Intelligent Technology Co., Ltd.) is composed of an O3 generating device and a CO2 cell incubator. An O3 generation monitoring system monitors the O3 concentration in the incubator in real time and controls the generation flow, so that the O3 concentration in the incubator is maintained within the range of ±0.03 ppm of the set value. Before each exposure, a pump suction type O3 detector is used for concentration calibration.

[0027] The application discloses an O3 gas-liquid exposure cell method based on a Transwell chamber. Figure 1 The application discloses an O3 gas-liquid exposure cell method based on a Transwell chamber. A certain number of cells are inoculated into the Transwell chamber to grow a monolayer, ensuring that the cells can be in full contact with the gas. Culture solution is added to the lower six-well plate. After the cells adhere and grow to about 70-80%, the culture solution in the chamber is aspirated. The cells are cultured in a normal CO2 cell incubator for a certain period of time as a control group. The cells are cultured in a cell incubator with O3 generation and real-time monitoring functions for the same period of time to realize O3 gas-liquid exposure of the cells in vitro. Compared with the control group, the changes in the cell morphology and key indicators of oxidative stress after O3 gas-liquid exposure are observed.

[0028] The raw materials used in the examples are as follows: The Transwell chamber is purchased from Corning Incorporated.

[0029] Example 1: An O3 gas-liquid exposure cell method based on a Transwell chamber 1. An O3 gas-liquid exposure method for mouse alveolar epithelial MLE12 cells based on a Transwell chamber, comprising the following steps: (1) Cell inoculation: 1 mL of 1×10 5 MLE12 cells / mL is inoculated into a Transwell chamber (upper chamber), and 2 mL of RPMI-1640 complete culture solution is added to the upper and lower chambers. After the cells adhere, the cells are cultured in a normal cell incubator (37 ℃, 5% CO2) for 36 h. (2) O3 gas-liquid exposure: After culture, the culture solution in the upper chamber was aspirated, the culture solution in the lower chamber was reserved and the porous membrane in the upper chamber was contacted with the culture solution, and the O3 exposure cell culture box (with the ordinary cell culture box as a control) was put in, treated with 0.4 ppm O3 for 3 h, and the O3 contaminated cells were obtained, which could be further observed and analyzed for cell damage.

[0030] 2. O3 contaminated MLE12 cell damage detection (1) Cell morphology After O3 gas-liquid exposure, the cell morphology and viability were observed under an optical microscope.

[0031] The results are shown in Table 1. Figure 2 As shown in Table 1, the MLE12 cells in the control group maintained normal spindle structure and were closely connected; compared with the control group, the number of MLE12 cells in the 0.4 ppm O3 gas-liquid exposure group was reduced, the morphology was elongated, the volume was reduced, and the connection was not close; it could be seen that O3 gas-liquid exposure affected the morphology of MLE12 cells.

[0032] (2) Intracellular ROS After the cells were washed with PBS, 500 μL of 1:1000 DCFH-DA diluent was added to each chamber, and incubated in the dark for 30 min, then digested with 500 μL of trypsin, terminated with 500 μL of complete culture medium, centrifuged at 1000 g for 3 min, the supernatant was discarded, and 1 mL of serum-free medium and PBS were used to resuspend and centrifuge at 1,000 g for 3 min, 200 μL of PBS was added to each tube, counted using a cell counter, and added to 100 μL per well of a 96-well plate, and the total fluorescence intensity was recorded using a fluorescence microplate reader with an excitation wavelength of 488 nm and an emission wavelength of 525 nm. Finally, the relative fluorescence intensity of each cell represented the ROS level.

[0033] The results are shown in Table 2. Figure 3 As shown in Table 2, compared with the control group, different concentrations of O3 gas-liquid exposure caused a significant increase in ROS levels in MLE12 cells, and had a dose-effect relationship.

[0034] (3) Intracellular GSH Cell pretreatment: after the end of the cell experiment, 20 μL of RIPA was added to each chamber, and lysed on ice for 20 min, the cells were scraped with a cell scraper, centrifuged at 12000 g for 30 min, and the supernatant was taken; Protein concentration determination: BCA method was used to determine the protein concentration, standard curve hole was added with 1 μg / μL protein standard 0, 1, 2, 4, 8, 12, 16, 20 μL, 1 μL of the protein to be determined was added to the determination hole, physiological saline was added to each hole to make up to 20 μL and 200 μL of BCA working solution was added, 37℃ incubation for 30 min, determination by enzyme labeling instrument.

[0035] GSH concentration determination: standard holes, determination holes and determination blank holes were set, the reaction system was prepared respectively, and the absorbance value at 405 nm was determined by using the enzyme labeling instrument after standing for 5 min, and the GSH content was calculated.

[0036] The results are shown in Figure 3 B, compared with the control group, different concentrations of O3 gas-liquid exposure caused a significant decrease in GSH level in MLE12 cells.

[0037] (4) SOD detection Cell pretreatment: after the end of the cell experiment, 20 μL RIPA was added to each chamber and lysed on ice for 20 min, the cells were scraped with a cell scraper and centrifuged at 12000 g for 30 min, and the supernatant was taken; Protein concentration determination: BCA method was used to determine the protein concentration, standard curve hole was added with 1 μg / μL protein standard 0, 1, 2, 4, 8, 12, 16, 20 μL, 1 μL of the protein to be determined was added to the determination hole, physiological saline was added to each hole to make up to 20 μL and 200 μL of BCA working solution was added, 37℃ incubation for 30 min, determination by enzyme labeling instrument.

[0038] SOD pre-experiment: the sample was diluted with RIPA to 1, 0.5, 0.1, 0.05 μg / μL for determination, control holes, control blank holes, determination holes and determination blank holes were set, the reaction system was prepared respectively, and after mixing, it was incubated at 37℃ for 20 min, the absorbance value at 450 nm was determined by using the enzyme labeling instrument, and the sample concentration with SOD inhibition rate of 40~60% (0.5 μg / μL) was selected as the subsequent experimental concentration.

[0039] SOD determination: all samples were diluted with RIPA to 0.5 μg / μL, and the SOD value was determined according to the above experiment, and the SOD inhibition rate and activity were calculated combined with the sample protein concentration.

[0040] As shown in Figure 3 C, compared with the control group, different concentrations of O3 gas-liquid exposure caused a significant increase in SOD level in MLE12 cells.

[0041] Example 2: A method for O3 gas-liquid exposure of cells based on Transwell chamber Using human bronchial epithelial cells Beas-2B as the detection object, using O3 of different concentrations (0.1, 0.2, 0.4 or 0.8 ppm) for 3 h, and the rest of the steps are consistent with Example 1, the damage of O3 to human bronchial epithelial Beas-2B cells is detected.

[0042] The cell morphology is shown in Figure 4 The control group Beas-2B cells maintain normal spindle structure and are closely connected; compared with the control group, the number of cells in the 0.1, 0.2, 0.4 and 0.8 ppm O3 gas-liquid exposure groups gradually decreases, the cell morphology gradually changes, the cell volume decreases, and the connection is not tight; the cell structure in the 0.8 ppm O3 gas-liquid exposure group is basically lost, and a large number of cells are detached and dispersed.

[0043] Therefore, 0, 0.1, 0.2 and 0.4 ppm are more appropriate doses for studying respiratory damage caused by O3 exposure by O3 gas-liquid exposure Beas-2B cells.

[0044] The ROS, GSH and SOD detection results are shown in Figure 5 Compared with the control group, the ROS level in the 0.4 ppm O3 gas-liquid exposure group Beas-2B cells is significantly increased, the GSH content is significantly decreased, and the SOD activity in the 0.2 and 0.4 ppm O3 gas-liquid exposure groups Beas-2B cells is significantly decreased. It can be seen that 0.4 ppm O3 gas-liquid exposure causes oxidative stress in Beas-2B cells.

[0045] Comparative Example 1: Conventional single-concentration O3 gas-liquid interface exposure method 1. Taking the single-concentration O3 gas-liquid interface exposure method as an example, conventional O3 cell exposure requires the purchase of a single-concentration cell gas-liquid interface exposure device (Beijing Huironghe, HRH-CES1332), which requires a large space, high cost, high difficulty in operation, high sterility requirement of the experimental environment, and only 3 planes with a diameter of 24 mm can be exposed to each dose. Based on the O3 gas-liquid exposure cell method of the Transwell chamber, the number of cells exposed can be arbitrarily selected according to the experimental requirements.

[0046] 2. Taking the O3 immersion direct ventilation exposure method as an example (1) Cell inoculation: Take 1 mL of 1×10 5 cells / mL of MLE12 cells, inoculate in a Transwell chamber, and add 2 mL of RPMI-1640 complete culture medium to the upper and lower chambers; place in a normal cell culture incubator (37 ℃, 5% CO2) for culture until the cells adhere; (2) O3 immersion direct ventilation exposure: Then, the O3-exposed cells were put into the O3-exposed cell incubator (with the normal cell incubator as a control) and treated with 0, 0.25, 0.5, 2 or 2 ppm O3 for 0, 6, 12, 24 or 48 h to obtain O3-exposed cells, which were further observed and analyzed for cell damage.

[0047] (3) Morphological observation of O3-exposed MLE12 cells: After O3 immersion direct ventilation exposure, the cell morphology and viability were observed under an optical microscope.

[0048] The results are shown in Table 1. Figure 6 As shown in Table 1, the MLE12 cells in the control group (0 h) maintained normal spindle structure and were closely connected; compared with the control group, 0.25 and 0.5 ppm O3 exposure for different time did not significantly change the morphology and number of MLE12 cells, while the number of MLE12 cells in the 1 and 2 ppm O3 exposure groups decreased significantly, the volume decreased, and the connection was not tight with the increase of exposure time.

[0049] Therefore, compared with the traditional single-concentration O3 gas-liquid interface exposure method and the O3 immersion direct ventilation exposure method, the O3 gas-liquid exposure method is more sensitive and efficient for cells.

[0050] Example 3: Screening of biomarkers for O3 exposure The O3 gas-liquid exposure method based on the Transwell chamber for screening biomarkers for O3 exposure includes the following steps: (1) Cell inoculation: 1 mL of 1×10 5 cells / mL of MLE12 cells were inoculated into the Transwell chamber (upper chamber), and 2 mL of RPMI-1640 complete culture solution was added to the upper and lower chambers; after the cells adhered, the culture was continued for 36 h in a normal cell incubator (37°C, 5% CO2); (2) O3 gas-liquid exposure: After the culture, the culture solution in the upper chamber was removed, the culture solution in the lower chamber was retained, and the multi-well membrane of the upper chamber was exposed to the culture solution; the O3-exposed cells were obtained by placing the cells into the O3-exposed cell incubator (with the normal cell incubator as a control) and treating with 0.4 ppm O3 for 3 h; (3) Further detection of the expression of genes and proteins in cells to screen biomarkers for O3 exposure.

[0051] Example 4: Screening of drugs for alleviating O3 exposure damage The O3 gas-liquid exposure method based on the Transwell chamber for screening drugs for alleviating O3 exposure damage includes the following steps: (1) Cell inoculation: Take 1 mL 1x10 5 6 cells / mL, inoculate into Transwell chamber (upper chamber), add 2 mL RPMI-1640 complete culture solution to the upper and lower chambers respectively; place in a general cell culture box (37℃, 5% CO2) until the cells adhere, then add the screening drug and continue to culture for 36 h; (2) O3 gas-liquid exposure: After culture, discard the culture solution in the upper chamber, retain the culture solution in the lower chamber and make the porous membrane of the upper chamber contact the culture solution, place in an O3 exposure cell culture box (use placement in a general cell culture box as a control), treat with 0.4 ppm O3 for 3 h, obtain O3-damaged cells; further observe and analyze the cell damage, and screen to obtain a drug that can alleviate O3 exposure damage.

[0052] Although the present application has been disclosed with the preferred embodiments as above, it is not intended to limit the present application, and any person skilled in the art can make various modifications and modifications without departing from the spirit and scope of the present application, therefore the protection scope of the present application should be defined by the claims.

Claims

1. A method for ozone-liquid cell treatment based on Transwell chambers, characterized in that, Including the following steps: (1) Seed cells in a Transwell chamber, add cell culture medium to the lower chamber, and incubate in a cell culture incubator; (2) After the cells adhere to the wall, remove the culture medium in the upper chamber, retain the culture medium in the lower chamber and contact the porous membrane of the Transwell chamber with the culture medium; place the Transwell chamber under ozone conditions.

2. The method according to claim 1, characterized in that, In step (1), the number of cells seeded is 1~3×10⁻⁶. 5 indivual.

3. The method according to claim 1, characterized in that, The cells used in step (1) include mouse alveolar epithelial cells MLE12, human bronchial epithelial cells Beas-2B, and mouse mononuclear macrophages RAW264.

7.

4. The method according to claim 1, characterized in that, In step (1), the culture medium added to the lower chamber is the culture medium required for cell inoculation, including Gibco RPMI 1640 medium and Gibco DMEM medium.

5. The method according to claim 1, characterized in that, In step (2), the ozone treatment is carried out at an ozone concentration of 0.1~0.8 ppm for 1~5 h.

6. The method according to claim 5, characterized in that, In step (2), the ozone treatment is carried out at an ozone concentration of 0.1~0.6 ppm for 1~5 h.

7. The method according to claim 6, characterized in that, In step (2), the ozone treatment is carried out at an ozone concentration of 0.2~0.4 ppm for 2~4 h.

8. The method according to claim 1, characterized in that, After treatment under ozone conditions in step (2), the damage status of cells under ozone conditions is evaluated by observing cell morphology or detecting the expression levels of ROS, GSH and SOD in cells.

9. The application of the method according to any one of claims 1 to 8 in screening biomarkers of O3 exposure.

10. The use of the method according to any one of claims 1 to 8 in screening drugs to alleviate O3 exposure damage.

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