A calibration method for ozone gas-liquid interface exposure concentration of in vitro cell experiment

Abstract

The present application relates to the technical field of environmental chemistry, and discloses a calibration method for ozone gas-liquid interface exposure concentration based on a tilting rotation mode for in-vitro cell experiments, which monitors and calibrates the actual effect concentration of ozone by using a chemical method, and can correct the problem of low actual effect concentration caused by a culture dish cover. The gas-liquid interface exposure system based on the tilting rotation mode is easy to build and low in cost, and can expose a large number of cells at a time. In combination with the calibration technology, the calibrated actual effect concentration is more accurate for in-vitro cell toxicity experiments.

Description

Technical Field

[0001] This invention relates to the field of environmental chemistry technology, specifically to a calibration method for the concentration of ozone-liquid interface exposure in in vitro cell experiments. Background Technology

[0002] Epidemiological surveys have found a correlation between ozone pollution and the incidence of respiratory diseases such as asthma and chronic obstructive pulmonary disease. Research on the toxic effects and mechanisms of ozone on the respiratory system is of significant reference value for the formulation of air quality standards and the protection of the respiratory system during human activities (or exercise). In vivo exposure experiments using animals are time-consuming and costly, and raise ethical concerns. In vitro exposure experiments using cells can significantly shorten the experimental period, reduce the use of experimental animals, lower experimental costs, and facilitate rapid and accurate assessment of the toxic effects of pollutants, laying the foundation for further in-depth research on their toxicological mechanisms.

[0003] Traditional cell exposure experiments employ immersion exposure, where pollutants are added to the culture medium and co-cultured with cells to study the toxicological effects of the pollutants on the cells. However, in the field of inhalation toxicology research, traditional immersion culture cannot simulate the gas-liquid interface exposure process in real-world scenarios. For studying the toxic effects of air pollutants (particulate matter, NOx, CO, SO2, O3, etc.) on the lungs, if traditional immersion culture is used, the air pollutants do not directly contact the cells but rather dissolve in the culture medium, indirectly affecting the cells. This cannot accurately assess the direct effects of pollutants on cells. Therefore, studying the toxicity of air pollutants to the lungs first requires establishing a gas-liquid interface exposure system that can simulate real-world scenarios.

[0004] Currently, there are two main types of gas-liquid interface exposure systems that have been developed: One type is the commercially available air-liquid interface (ALI) device. Its principle is to seed cells in the upper chamber of a Transwell chamber, with culture medium added to the lower chamber. This allows the lower part of the cells in the upper chamber to contact the culture medium, while the upper part is directly exposed to the gas phase. Although this method can simulate real air-liquid interface exposure well, the device is expensive and has a low throughput, resulting in a very limited number of cells that can be cultured each time. This limits its application to experiments that require obtaining a large number of cell samples.

[0005] Another simple gas-liquid interface exposure method (hereinafter referred to as the tilt-rotation method) involves placing a slowly rotating tilted platform (or shaker) at a constant speed (tilt angle ~10°) inside a cell culture incubator. Culture dishes are placed on this platform (or shaker), and the amount of culture medium added is controlled. As the tilted platform rotates, the cells in the dish periodically alternate between being covered by the culture medium and exposed to gas. This can simulate gas-liquid interface exposure to some extent, and the system is easy to build, low in cost, and can expose a large number of cells at once. Researchers have already used this method in in vitro ozone exposure experiments on cells. However, this method also has a drawback: to ensure aseptic culture of the cells, the culture dishes must be covered, and the presence of the cover affects the contact between ozone and cells, weakening the ozone's effect on the cells. Therefore, it is necessary to monitor and calibrate the actual effect of this gas-liquid interface exposure method. Summary of the Invention

[0006] (a) Technical problems to be solved To address the shortcomings of existing technologies, this invention provides a calibration method for ozone-liquid interface exposure concentration in in vitro cell experiments. This method corrects the problem of low actual effect concentration caused by the culture dish lid. The calibrated actual effect concentration is more accurate for in vitro cell toxicology experiments. Furthermore, it offers the advantages of easy system setup, low cost, and the ability to expose a large number of cells at once. (II) Technical Solution To correct the problem of low actual effect concentration caused by the petri dish lid, the present invention provides the following technical solution: a calibration method for ozone gas-liquid interface exposure concentration in in vitro cell experiments, comprising the following steps; S1: Prepare the chemical reagents required for the actual effect concentration detection; S2: Set up a blank control group, a covered exposure group with a specific concentration of ozone, and an uncovered exposure group with a specific concentration of ozone. Detect the oxidation effect of each group after exposure for different durations (30 min, 1 h, 2 h, 3 h, 4 h, 5 h...). Set up 3 replicates for each sampling time point in each group. S3: Calculate and calibrate the actual ozone effect concentration using a formula.

[0007] Preferably, the raw materials include K2HPO4, KH2PO4, EDTA, DTT, DTNB, Na2HPO4, pure water, HCl and KOH, and the chemical reagents required in step S1 are 0.1 M pH 7.3 potassium phosphate buffer, 1 mM DTT solution and 10 mM DTNB solution.

[0008] Preferably, the preparation method of the 0.1 M pH 7.3 potassium phosphate buffer is as follows: (1) Weigh 14.9712 g K2HPO4·3H2O, 4.682 g KH2PO4 and 292.24 mg EDTA respectively, add them to 800 mL of pure water and stir until completely dissolved; (2) Adjust the pH to 7 using KOH solution; (3) Add pure water to make up to 1 L.

[0009] Preferably, the method for preparing the 1 mM DTT solution is as follows: (1) Weigh 154.251 mg of DTT powder and dissolve it in pure water to prepare 1 mL of 1 M DTT (1000X) stock solution (this stock solution can be stored at -20 ℃ in the dark for more than 2 years). (2) Take 100 μL of the above stock solution and dilute it 1000 times with pure water to prepare 100 mL of 1 mM DTT. Store at 4 ℃ in the dark for a short period of time. (3) Preparation of 50 mM Na2HPO4 (pH 7.0) (taking 10 mL as an example).

[0010] Preferably, the method for preparing the 10 mM DTNB solution is as follows: (1) Weigh 39.635 mg DTNB and dissolve it in 10 mL of the above 50 mM Na2HPO4 (pH 7.0) solution; (2) Sterilize by filtration with a 0.22 μm filter membrane (do not use high temperature and high pressure), and store the solution at 4 ℃ in the dark for 2 months.

[0011] Preferably, the specific steps of step S2 are as follows: (1) Prepare a mixed solution at a ratio of 1 mL of 0.1 M potassium phosphate buffer (pH 7.3) + 100 μL of 1 mM DTT per dish; (2) Add the mixed solution described in S(1) to a 3.5 cm cell culture dish at a volume of 1 mL; (the volume of the mixed solution added can be adjusted according to the size of the culture dish so that the liquid just covers half of the bottom area of ​​the dish when the culture dish is placed on a tilted platform). (3) Transfer the blank control group dish containing the mixed solution to a conventional cell culture incubator for incubation, and transfer the exposure group dish containing the mixed solution to the tilted rotating platform inside the exposure chamber. Close the chamber door and begin exposure. (4) Take out 3 dishes at 30 min, 1 h, 2 h, 3 h, 4 h, 5 h... respectively, add pure water to make up to 1 mL, immediately add 50 μL 10 mM DTNB and mix well, and detect the absorbance of the solution at 412 nm wavelength within 2 h.

[0012] Preferably, the method for calculating and calibrating the actual ozone effect concentration in step S3 is as follows: The oxidation effect is represented by the amount of DTT consumed, and the specific calculation formula is as follows: ΔDTT=(OD t空白 -OD t ) / OD0*DTT0(Formula ①) Wherein; OD0; is the initial (0 min) background absorbance after DTT potassium phosphate buffer color development. OD t Blank; absorbance values ​​of the blank control group after DTT potassium phosphate buffer staining at different time points. OD t Absorbance values ​​of each exposure group after DTT potassium phosphate buffer staining at different time points. DTT0; Initial concentration of DTT (nmol). To detect the actual oxidation effect of ozone at different set concentrations, the initial amount of DTT can be adjusted to ensure sufficient DTT throughout the detection process. Actual ozone concentration = (ΔDTT with cover / ΔDTT without cover) * ozone set concentration (Equation ②) The calculated actual ozone effect concentration is the calibrated actual effect concentration.

[0013] (III) Beneficial Effects Compared with the prior art, the present invention provides a calibration method for ozone-liquid interface exposure concentration in in vitro cell experiments, which has the following beneficial effects: In summary, this method uses chemical methods to monitor and calibrate the actual effective concentration of ozone, correcting the problem of low actual effective concentration caused by the petri dish lid. Compared with existing gas-liquid interface exposure technologies based on Transwell chambers, the gas-liquid interface exposure system based on tilting and rotation is easier to build, lower in cost, and can expose a large number of cells at once. Combined with this calibration technology, the calibrated actual effective concentration is more accurate for in vitro cytotoxicology experiments. Attached Figure Description

[0014] Figure 1 This is a schematic diagram showing the consumption of DTT under different exposure durations in a calibration method for ozone-liquid interface exposure concentration for in vitro cell experiments according to the present invention. Figure 2 This is a schematic diagram showing the oxidation effect ratio of covered / uncovered under different exposure durations under a calibration method for ozone-liquid interface exposure concentration in in vitro cell experiments according to the present invention. Figure 3This is a schematic diagram of the intracellular reactive oxygen species level of BEAS-2B cells after exposure, which is a calibration method for ozone gas-liquid interface exposure concentration in in vitro cell experiments according to the present invention. Figure 4 This is a schematic diagram of an immersion exposure method for calibrating the concentration of ozone at the liquid interface in in vitro cell experiments according to the present invention. Figure 5 This is a schematic diagram of a gas-liquid interface exposure device based on a Transwell chamber, which is a calibration method for ozone gas-liquid interface exposure concentration in in vitro cell experiments according to the present invention. Figure 6 This is a schematic diagram of tilted rotating gas-liquid interface exposure, which is a calibration method for ozone gas-liquid interface exposure concentration in in vitro cell experiments according to the present invention. Detailed Implementation

[0015] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0016] Example 1; Please refer to Tables 1-4; A calibration method for ozone-liquid interface exposure concentration in in vitro cell experiments, and preparation of chemical reagents required for actual effect concentration detection; The raw materials include K2HPO4, KH2PO4, EDTA, DTT, DTNB, Na2HPO4, pure water, HCl and KOH. The required chemical reagents are 0.1 M pH 7.3 potassium phosphate buffer, 1 mM DTT solution and 10 mM DTNB solution. (1) The preparation method of 0.1 M pH 7.3 potassium phosphate buffer is as follows (taking 1 L as an example). Table 1 Potassium phosphate buffer formulation 1) Add the reagents in Table 1 to 800 mL of pure water in sequence and stir until completely dissolved; 2) Adjust the pH to 7.3 using KOH solution; 3) Add pure water to bring the volume to 1 L.

[0017] (2) Preparation of 1 mM DTT solution (taking 100 mL as an example) Table 2 DTT solution formulation 1) Dissolve DTT powder in pure water according to Table 2 to prepare 1 mL of 1 M DTT (1000X) stock solution (this stock solution can be stored at -20℃ in the dark for more than 2 years). 2) Take 100 μL of the above stock solution and dilute it 1000 times with pure water to prepare 100 mL of 1 mM DTT. Store at 4 ℃ in the dark for a short period of time.

[0018] (3) Preparation of 50 mM Na2HPO4 (pH 7.0) (taking 10 mL as an example) Table 3. Na2HPO4 solution formulation 1) Weigh 179.07 mg of Na2HPO4·12H2O and prepare 10 mL of 50 mM Na2HPO4 solution; 2) Adjust the pH to 7.0 with HCl.

[0019] 3) Na2HPO4 can also be replaced with K2HPO4 here. Accordingly, the required amount needs to be calculated based on the molecular weight. (4) Preparation of 10 mM DTNB (taking 10 mL as an example) Table 4. Na2HPO4 solution formulation 1) Weigh 39.635 mg of DTNB and dissolve it in 10 mL of the 50 mM Na2HPO4 (pH 7.0) solution described in (3) (DTNB is slightly soluble in water, so a 50 mM Na2HPO4 (pH 7.0) solution is required as the solvent); 2) Sterilize by filtration through a 0.22 μm filter membrane (do not use high temperature or high pressure). The solution can be stored at 4 ℃ in the dark for 2 months.

[0020] Example 2; Please refer to the technical solution in this manual; set up a blank control group, a covered exposure group with a specific concentration of ozone, and an uncovered exposure group with a specific concentration of ozone. Detect the oxidation effect of each of the above groups after exposure for different durations (30 min, 1 h, 2 h, 3 h, 4 h, 5 h...). Set up 3 replicates for each sampling time point in each group. The specific steps of step S2 are as follows; (1) Prepare a mixed solution at a ratio of 1 mL of 0.1 M potassium phosphate buffer (pH 7.3) + 100 μL of 1 mM DTT per dish; (2) Add the mixed solution from S(1) to a 3.5 cm cell culture dish at a ratio of 1 mL; (the volume of the mixed solution added can be adjusted according to the size of the culture dish so that the liquid just covers half of the bottom area of ​​the dish when the culture dish is placed on a tilted platform). (3) Transfer the blank control group dish containing the mixed solution to a conventional cell culture incubator for incubation, and transfer the exposure group dish containing the mixed solution to the tilted rotating platform inside the exposure chamber. Close the chamber door and begin exposure. (4) Take out 3 dishes at 30 min, 1 h, 2 h, 3 h, 4 h, 5 h... respectively, add pure water to make up to 1 mL, immediately add 50 μL of 10 mM DTNB and mix well, and detect the absorbance of the solution at 412 nm wavelength within 2 h.

[0021] Example 3; Please refer to the technical solution in this manual; calculate and calibrate the actual ozone effect concentration using the formula; The oxidation effect is represented by the amount of DTT consumed, and the specific calculation formula is as follows: ΔDTT=(OD t空白 -OD t ) / OD0*DTT0(Formula ①) Wherein; OD0; is the initial (0 min) background absorbance after DTT potassium phosphate buffer color development. OD t空白 The absorbance values ​​of the blank control group after DTT potassium phosphate buffer staining at different time points. OD t Absorbance values ​​of each exposure group after DTT potassium phosphate buffer staining at different time points. DTT0; Initial concentration of DTT (nmol). To detect the actual oxidation effect of ozone at different set concentrations, the initial amount of DTT can be adjusted to ensure sufficient DTT throughout the detection process. Actual ozone concentration = (ΔDTT with cover / ΔDTT without cover) * ozone set concentration (Equation ②) The calculated actual ozone effect concentration is the calibrated actual effect concentration.

[0022] Example 4; Please see Figure 3The calibration of the actual effect concentration at a set concentration of 0.5 ppm was performed using the tilted rotating gas-liquid interface exposure system and oxidation effect detection method described in this manual, under the condition that the ozone set concentration was 0.5 ppm. The oxidation effect of the covered exposure group was greatly reduced, and the oxidation effect ratio between the covered group and the uncovered group was 35% to 43%. The actual ozone effect concentration calculated according to formulas ① and ② was 0.175 ppm to 0.215 ppm, which is comparable to the concentration of severe ozone pollution in the environment.

[0023] Example 5; Please see Figure 1-3 The effect of ozone exposure at a set concentration of 0.5 ppm on intracellular reactive oxygen species (ROS) levels in BEAS-2B cells was investigated. Using the tilted rotating gas-liquid interface exposure system described in this manual, human bronchial epithelial cells (BEAS-2B) were exposed to ozone at a set concentration of 0.5 ppm (simulating actual effect concentrations of 0.175 ppm to 0.215 ppm) for 8 hours. Intracellular ROS levels were then measured. The results showed that exposure to ozone at a set concentration of 0.5 ppm (simulating actual effect concentrations of 0.175 ppm to 0.215 ppm) for 8 hours significantly increased intracellular ROS levels in BEAS-2B cells.

[0024] Comparative Example 1: Please see Figure 4 Traditional cell exposure experiments employ immersion exposure, where pollutants are added to the culture medium and co-cultured with cells to study the toxicological effects of the pollutants on the cells. However, in the field of inhalation toxicology research, traditional immersion culture cannot simulate the gas-liquid interface exposure process in real-world scenarios, particularly for air pollutants (particulate matter, NO). x Studies on the toxic effects of air pollutants (such as CO, SO2, and O3) on the lungs using traditional immersion culture methods cannot accurately assess the direct effects of pollutants on cells because air pollutants do not come into direct contact with cells but rather dissolve in the culture medium.

[0025] Comparative Example 2: Please see Figure 5 Existing commercial gas-liquid interface exposure devices work by seeding cells in the upper chamber of a Transwell chamber and adding culture medium to the lower chamber. This allows the lower part of the cells in the upper chamber to contact the culture medium, while the upper part is directly exposed to the gas phase. Although this method can simulate real gas-liquid interface exposure well, the devices are expensive and have low throughput, resulting in a very limited number of cells that can be cultured each time. This limits their application to experiments that require a large number of cell samples.

[0026] Comparative Example 3: Please see Figure 6The existing simple gas-liquid interface exposure method (hereinafter referred to as the tilt-rotation method) involves placing a slowly rotating tilted platform (or shaker) with an angle of approximately 10° inside a cell culture incubator. Culture dishes are placed on this platform (or shaker), and the amount of culture medium added is controlled. As the tilted platform rotates, the cells in the dish periodically alternate between being covered by the culture medium and exposed to gas. This method can simulate gas-liquid interface exposure to some extent, and the system is easy to build, low in cost, and can expose a large number of cells at once. Researchers have already used it in in vitro ozone exposure experiments on cells. However, this method also has a drawback: to ensure aseptic culture of the cells, the culture dishes must be covered, and the presence of the cover affects the contact between ozone and cells, weakening the ozone's effect on the cells. Therefore, it is necessary to monitor and calibrate the actual effect of this gas-liquid interface exposure method.

[0027] In summary, this method uses chemical methods to monitor and calibrate the actual effective concentration of ozone, correcting the problem of low actual effective concentration caused by the petri dish lid. Compared with existing gas-liquid interface exposure technologies based on Transwell chambers, the gas-liquid interface exposure system based on tilting and rotation is easier to build, lower in cost, and can expose a large number of cells at once. Combined with this calibration technology, the calibrated actual effective concentration is more accurate for in vitro cytotoxicology experiments.

[0028] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0029] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A calibration method for ozone-liquid interface exposure concentration in in vitro cell experiments, characterized in that: Includes the following steps; S1: Prepare the chemical reagents required for the actual effect concentration detection; S2: Set up a blank control group, a covered exposure group with a specific concentration of ozone, and an uncovered exposure group with a specific concentration of ozone. Detect the oxidation effect of each of the above groups after exposure for different durations of 30 min, 1 h, 2 h, 3 h, 4 h, 5 h, or longer. Set up 3 replicates for each sampling time point in each group. S3: Calculate and calibrate the actual ozone effect concentration using a formula; The method includes the following raw materials: K2HPO4, KH2PO4, EDTA, DTT, DTNB, Na2HPO4, pure water, HCl and KOH. The chemical reagents required in step S1 are 0.1 M pH 7.3 potassium phosphate buffer, 1 mM DTT solution and 10 mM DTNB solution. The preparation method of 10 mL of the 10 mM DTNB solution is as follows: (1) Weigh 39.635 mg DTNB and dissolve it in 10 mL of 50 mM pH 7.0 Na2HPO4 solution; (2) Sterilize by filtration with a 0.22 μm filter membrane. Do not use high temperature or high pressure. Store the solution at 4 ℃ in the dark for 2 months. The specific steps of step S2 are as follows: (1) Prepare a mixed solution at a ratio of 1 mL of 0.1 M pH 7.3 potassium phosphate buffer + 100 μL of 1 mM DTT per dish; (2) Add 1 mL of the mixed solution to each 3.5 cm cell culture dish; when the culture dish is placed on a tilted platform, the liquid should just cover half of the bottom area of ​​the dish; (3) Transfer the blank control group dish containing the mixed solution to a conventional cell culture incubator for incubation, and transfer the exposure group dish containing the mixed solution to the tilted rotating platform inside the exposure chamber. Close the chamber door and begin exposure. (4) Take out 3 dishes at 30 min, 1 h, 2 h, 3 h, 4 h, 5 h or longer, add pure water to make up to 1 mL, immediately add 50 μL 10 mM DTNB and mix well. Detect the absorbance of the solution at 412 nm wavelength within 2 h. The method for calculating and calibrating the actual ozone effect concentration in step S3 is as follows: The oxidation effect is represented by the amount of DTT consumed, denoted as ΔDTT. The specific calculation formula is as follows: ΔDTT=(OD t空白 -OD t ) / OD0*DTT0 (Equation ①) in; OD0; initial absorbance after 0 min of DTT potassium phosphate buffer color development. OD t空白 The absorbance values ​​of the blank control group after DTT potassium phosphate buffer staining at different time points. OD t Absorbance values ​​of each exposure group after DTT potassium phosphate buffer staining at different time points. DTT0; the initial concentration of DTT, in nmol. The actual oxidation effect of ozone at different set concentrations was detected. By adjusting the initial amount of DTT, sufficient DTT was ensured throughout the detection process. Actual ozone concentration = (ΔDTT with cover / ΔDTT without cover) * ozone set concentration (Equation ②) The calculated actual ozone effect concentration is the calibrated actual effect concentration.

2. The calibration method for ozone-liquid interface exposure concentration in in vitro cell experiments according to claim 1, characterized in that: The preparation method of the 0.1 M pH 7.3 potassium phosphate buffer solution is as follows: (1) Weigh 14.9712 g K2HPO4·3H2O, 4.682 g KH2PO4 and 292.24 mg EDTA respectively, add them to 800 mL of pure water and stir until completely dissolved; (2) Adjust the pH to 7 using KOH solution; (3) Add pure water to make up to 1 L.

3. The calibration method for ozone-liquid interface exposure concentration in in vitro cell experiments according to claim 2, characterized in that: The preparation method of 100 mL of the 1 mM DTT solution is as follows: (1) Weigh 154.251 mg of DTT powder and dissolve it in pure water to prepare 1 mL of 1 M DTT stock solution. The stock solution should be stored at -20℃ in the dark for more than 2 years. (2) Take 100 μL of the above stock solution and dilute it 1000 times with pure water to prepare 100 mL of 1 mM DTT. Store at 4℃ away from light for a short period of time.

4. The calibration method for ozone-liquid interface exposure concentration in in vitro cell experiments according to claim 2, characterized in that: The method for preparing 10 mL of pH 7.0 Na2HPO4 solution is as follows: Weigh 179.07 mg of Na2HPO4·12H2O to prepare 10 mL of 50 mM Na2HPO4 solution, and adjust the pH to 7.0 with HCl. Na2HPO4 can also be replaced with K2HPO4. Accordingly, the required amount needs to be calculated based on the molecular weight.

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