CN110736696A - method for determining cigarette smoke cytotoxicity under exposure of gas-liquid interface - Google Patents

Abstract

The invention relates to the field of tobacco and tobacco product detection, in particular to a method for determining the cytotoxicity of cigarette smoke under gas-liquid interface exposure. The cytotoxicity caused by cigarette smoke is detected. When performing the gas-liquid interface exposure method, cells are seeded on an insert culture plate (also known as Transwell culture plate) at a density of 0.8-1.2*10 ⁶ cells/well; Cells were infected with flue gas at a flow rate of 8-12 mL/min. The detection method in the present invention can more objectively reflect the process of the human respiratory system contacting cigarette smoke, which not only improves the detection accuracy and sensitivity, but also reduces the detection time and is more convenient and quick. At the same time, the present invention can realize the evaluation of cytotoxicity of heat-not-burn cigarette and traditional cigarette smoke, and has wider coverage, simple operation and more comprehensive and reliable results than the prior art.

Description

A method for measuring cytotoxicity of cigarette smoke under exposure to gas-liquid interface

technical field

The invention relates to the field of tobacco and tobacco product detection, in particular to a method for measuring the cytotoxicity of cigarette smoke under exposure to a gas-liquid interface.

Background technique

Cytotoxicity test of cigarette smoke is one of the important means to evaluate the health effects of smoking. The cytotoxicity test mainly includes two steps: exposure of the test substance and detection of cell viability after exposure. Cigarette smoke exposure is the first step in measuring cytotoxicity. At present, cigarette smoke exposure mostly adopts two methods: smoke condensate and gas-liquid interface exposure in actual contact state. When the cells are exposed to the flue gas condensate, the flue gas condensate extracted by the solvent is in contact with the cells in the culture medium environment, and the flue gas composition is quite different in the solution and the aerosol, and the flue gas composition in the solution cannot truly reflect the exposure of the human body to smoke The real state of aerosol and the long exposure time. Air-liquid interface exposure exposure uses fresh smoke to directly contact the cells located in the upper part of the culture medium, which can truly simulate the state of the human respiratory system exposed to smoke, and more truly and comprehensively reflect the biological effects of cigarette smoke. In addition, during the exposure of the gas-liquid interface, the cell reaction is more sensitive than that of the flue gas condensate exposure, which can quickly complete the exposure process and shorten the entire experimental time.

In 2002, the International Tobacco Scientific Research Cooperation Center recommended the use of neutral red test method to determine cytotoxicity, but this method has complicated operation steps and involves multiple cleanings, which is easy to introduce errors. The thiazolyl blue method, referred to as "MTT method", is a test method different from neutral red. This detection method does not require fixation and washing of cells. After exposure to cigarette smoke and cell incubation, cells can be directly detected. Therefore, the loss of cells caused by multiple washing steps can be avoided.

With increasingly stringent tobacco regulatory legislation and in-depth smoking and health research, tobacco companies are striving to seek new lower-risk tobacco products as companies' future alternatives to traditional cigarettes. As a new type of tobacco product, heat-not-burn cigarettes release far less harmful components in the smoke than traditional cigarettes. However, for the cytotoxicity detection of heat-not-burn cigarette smoke, most of them use the exposure method of smoke condensate, and no gas-liquid interface exposure method has been found.

At present, there is no report on the combination of air-liquid interface exposure and thiazole blue to detect the cytotoxicity caused by cigarette smoke. In addition, there is no report on the combination of the above methods to detect the cytotoxicity of heat-not-burn tobacco products.

SUMMARY OF THE INVENTION

In order to solve the above technical problems, the present invention provides a method that can improve the sensitivity and accuracy of detection, and at the same time simply and quickly measure the cytotoxicity of cigarette smoke under exposure to the gas-liquid interface. The specific technical scheme is as follows:

Combined air-liquid interface exposure method and thiazolyl blue method (also known as MTT method) to detect cytotoxicity induced by cigarette smoke. This method is suitable for both traditional cigarette products and new tobacco product smoke cytotoxicity detection.

During the air-liquid interface exposure method, cells were seeded on an insert culture plate (also known as Transwell culture plate) at a density of 0.8-1.2*10 ⁶ cells/well; smoke at a flow rate of 8-12 mL/min Gas to infect cells.

The combined detection method of air-liquid interface exposure and thiazole blue takes fresh smoke as the test object, which can better reflect the process of human respiratory system exposure to cigarette smoke; cells are more sensitive to smoke during the exposure process, thus shortening the duration of exposure to cigarette smoke. The exposure time of the test substance; during the detection process, there is no need to wash the cells, which avoids the loss of cells and improves the detection accuracy.

However, although the air-liquid interface exposure method can enhance the sensitivity of cells to smoke, it is prone to cell death due to non-smoke causes (showing that the air control group also has an excess of cell death) or reduced cell sensitivity (It was manifested that the number of dead cells in the smoke treatment group was significantly less than expected), which made it difficult to balance the sensitivity and accuracy of the detection. In this regard, the present invention finds that the seeding density of cells and the flow rate of flue gas treatment have a greater impact on the above problems, and after many attempts, the optimization has been obtained: when the seeding density of cells and the flow rate of flue gas treatment are at the same time When it is within the above-mentioned limited range, it is beneficial to take into account the sensitivity and accuracy of smoke detection.

Preferably, the inoculation amount of the cells is 1.0*10 ⁶ cells/well, and the flow rate of the flue gas is 10 mL/min.

In order to further optimize the detection of flue gas, the present invention also optimizes other conditions to obtain the following conditions:

Preferably, during the poisoning treatment, the distance between the fume outlet and the permeable membrane of the inserted culture plate is 1.4-1.6 mm; preferably 1.5 mm.

Preferably, the exposure dose of smoke without dilution air is set as 100%, and different exposure doses of smoke are obtained by sequentially diluting the smoke by adjusting the flow rate of the dilution air.

Preferably, the suction mode is the ISO 3308:2012 suction mode or the Canadian deep suction mode; preferably the ISO 3308:2012 suction mode.

Preferably, the present application also optimizes the smoke exposure dose for the smoking mode. When the smoking mode is the ISO 3308:2012 smoking mode, the exposure time is 0.5-1.5 h; preferably 1 h.

Preferably, the cell is mouse mononuclear macrophage RAW264.7.

Preferably, the insert culture plate is a 24mm insert culture plate.

Preferably, during cell culture, 1.8-2.2mL (preferably 2mL) growth medium is added to the lower chamber, and 1.4-1.6mL (preferably 1.5mL) growth medium is added to the upper chamber;

Preferably, the cells are cultured for 24 hours before being exposed to the virus.

Preferably, when performing the thiazole blue method, the concentration of the MTT solution is 0.45-0.55 mg/mL, preferably 0.5 mg/mL.

It is found in the present invention that in the technical solution of the present invention (especially after setting a specific cell seeding number and a specific suction mode), setting the concentration of the MTT solution to the above range is more conducive to improving the detection effect.

Preferably, the specific operation of the thiazolyl blue method includes: adding 0.8-1.2 mL (preferably 1 mL) of 0.45-0.55 mg/mL mg/mL MTT solution to the insert-type culture plate, and in 5% CO ₂ , Incubate at 37°C for 2.5-3.5h (preferably 3h);

Preferably, after the incubation, remove the MTT solution in the chamber of the insert culture plate, add 0.8-1.2 mL of dimethyl sulfoxide, and gently shake for 4-6 min.

Preferably, a clean air exposure chamber is set up in each test, and clean air is passed into it, as an air control group; a smoke exposure chamber is set up, and smoke gas is passed into it, as an exposure group; and dimethyl sulfoxide is set in the absorbance detection. as blank group;

Cell survival rate=(absorbance of exposure group-absorbance of blank group)/(absorbance of air control group-absorbance of blank group)*100%.

Preferably, each exposure group is set to consist of three parallel chambers, enabling three parallel experiments for each exposure dose point.

The above parallel experiments further ensure the accuracy of the test results.

As a preferred technical solution of the present invention, the method comprises the following steps:

(1) Cell seeding: Select mouse mononuclear macrophages RAW264.7 in logarithmic growth phase and seed them on a 24mm insert culture plate at a density of 1*10 ⁶ cells/well, in which 1.8-2.2 mL is added to the lower chamber Growth medium, add 1.4-1.6 mL of growth medium to the upper chamber, and cultivate for 24 hours;

(2) Cigarette smoke exposure: After 24 hours of cell culture, the insert culture plate was moved to the exposure chamber, wherein the upper layer of the microporous membrane of the Transwell chamber was the smoke environment, the lower layer was the exposure culture medium, and the cells were in the gas-liquid state. Junction;

Set the distance between the flue gas outlet in the exposure chamber and the permeable membrane of the insert culture plate to be 1.4-1.6 mm, and the gas flow rate to be 8-12 mL/min;

The selected suction mode is ISO 3308:2012 suction mode, and the exposure time is 0.5-1.5h;

Set the exposure dose of flue gas without dilution air to 100%, and by adjusting the flow rate of dilution air, dilute the flue gas in turn to obtain different exposure doses of flue gas;

In each test, a clean air exposure chamber was set up, and clean air was introduced, as the air control group;

(3) MTT cytotoxicity test: after the exposure to the flue gas, the chamber on the insert culture plate was taken out from the exposure unit, and 0.8-1.2 mL of 0.45-0.55 mg/mL MTT solution was added under 5% CO ₂ , Incubate at 37°C for 2.5-3.5h;

Centrifuge to remove excess MTT solution in the upper chamber, add 0.8-1.2 mL of dimethyl sulfoxide, and shake gently for 4-6 min;

Set dimethyl sulfoxide as a blank group, and use a microplate reader to measure the absorbance at 490 nm of the blank group, the air control group and the exposure group respectively;

(4) Results and analysis: The cytotoxicity is expressed by the cell survival rate. The higher the cell survival rate, the lower the cytotoxicity of the flue gas, and vice versa, the higher the cytotoxicity of the flue gas;

Cell survival rate=(absorbance of exposure group-absorbance of blank group)/(absorbance of air control group-absorbance of blank group)*100%.

Preferably, the growth medium and exposure medium are DEME-H basal medium plus 10% fetal bovine serum.

Preferably, the preparation method of the PBS (0.01M phosphate buffer solution) is as follows: take 0.2gKCl, 0.2gKH2PO4, 8gNaCl, _{2g Na2HPO4 · 12H20} , add ultrapure water to 1L, and measure the pH to be 7.2 , autoclaved.

Preferably, the preparation method of the MTT solution is as follows: Weigh 0.250 g MTT, add 50 ml of PBS, dissolve and filter through a 0.22 μm microporous membrane, divide the package, and store at -20°C.

Preferably, a fully automatic smoking machine is used, and various smoking modes such as ISO 3308:2012 smoking mode and Canadian deep smoking mode are used to continuously generate smoke and uniformly deliver it to the smoke exposure unit.

The present invention further provides the application of the above method in the cytotoxicity test of traditional cigarette or heat-not-burn cigarette smoke.

The beneficial effects of the present invention are as follows:

The detection method in the present invention can more objectively reflect the process of the human respiratory system contacting cigarette smoke, which not only improves the detection accuracy and sensitivity, but also reduces the detection time and is more convenient and quick.

At the same time, the present invention can realize the evaluation of cytotoxicity of heat-not-burn cigarette and traditional cigarette smoke, and has wider coverage, simple operation and more comprehensive and reliable results than the prior art.

Detailed ways

The following examples are intended to illustrate the present invention, but not to limit the scope of the present invention.

The reagents and equipment used in the following examples are as follows:

(1) Instruments and devices used

Carbon dioxide incubator (SANYO Corporation, Japan), microplate reader (Bio-tek Corporation, USA), air-liquid interface exposure system (Beijing Huironghe Technology Co., Ltd., China), automatic smoking machine (Beijing Huironghe Technology Co., Ltd., China) Co., Ltd., China), automatic cell counter (ThermoFish Scientific, USA), inverted microscope (OLYMPUS, Japan), ultra-clean workbench (Suzhou Antai Air Technology Co., Ltd., China), 24mm insert 6-well culture plate (Transwell plate) (Corning, USA).

(2) Reagents and materials used

Mouse mononuclear macrophage RAW 264.7 (ATCC cell bank, USA), DMEM-H basal medium (GENVIEW, Germany), Fetal Bovine Serum (FBS) (AusGeneX, Australia), 3R4F cigarette ( University of Kentucky reference cigarette, USA), 1mg cigarette (box labeled 1mg tar blended cigarette, China), heat not burn cigarette (original type, purchased from Japan), MTT (3-(4,5-dimethyl-2 -thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide, 3-(4,5-dimethylthiazole-2)-2,5-diphenyltetrazolium bromide) (purity ≥99.0% , SIGMA-ALDRICH, USA), trypan blue (purity ≥99.0%, SIGMA-ALDRICH, USA), dimethyl sulfoxide (purity ≥99.0%, Sinopharm Chemical Reagent Co., Ltd., China).

Growth medium (DEME-H basal medium plus 10% FBS), exposure medium (DEME-H basal medium plus 10% FBS, 0.01M phosphate buffer solution (0.2gKCl, 0.2gKH2PO4, 8gNaCl, _{2g Na2} HPO ₄ ·12H ₂ 0, add ultrapure water to 1L, measure pH to 7.2, autoclave), MTT solution (weigh 0.250g MTT, add 50ml PBS, dissolve and filter with 0.22μm microporous membrane, divide into packages, -20°C storage).

Example 1 In vitro cytotoxicity evaluation of heat-not-burn cigarette smoke

Specifically include the following steps:

(1) Cell inoculation culture

The mouse mononuclear macrophage RAW264.7 in logarithmic growth phase was selected and seeded in a 24mm insert culture plate (Transwell culture plate) at a density of ¹ *106/well, and 2mL growth medium was added to the lower chamber, 1.5 mL of growth medium was added to the upper chamber and incubated for 24 h.

(2) Cigarette smoke poisoning

After the cells were cultured for 24 hours, the Transwell culture plate was moved to the exposure chamber, where the upper layer of the microporous membrane of the Transwell chamber was in a flue gas environment, and the lower layer was the exposed culture medium, and the cells were at the gas-liquid junction.

Using a fully automatic smoking machine, the heat-not-burn cigarettes were smoked according to the ISO 3308:2012 smoking mode (35mL/60s/2s), and the continuously generated smoke was evenly delivered to the smoke exposure unit. The distance between the bell mouth in the exposure chamber and the permeable membrane of the Transwell culture plate is 1.5 mm. The exposure chamber gas flow rate was set to 10 mL/min via a negative pressure pump and a microflow controller. Set the exposure dose of smoke without applying dilution air to 100%, by adjusting the flow rate of dilution air, by adjusting the flow rate of dilution air, set the exposure dose of smoke to 10%, 20%, 40%, 60% and 80% , smoke exposure for 1h. In each test, a clean air exposure chamber was set up, and clean air was introduced to serve as an air control group; a smoke exposure chamber was set up, and smoke gas was introduced to serve as an exposure group; Three parallel experiments at each exposure dose point.

(3) MTT cytotoxicity test

After the smoke exposure, the chamber on the Transwell plate was taken out from the exposure unit, 1 mL of 0.5 mg/mL MTT solution was added, 5% CO ₂ and a 37 °C incubator for 3 h. The excess MTT solution in the upper chamber was removed by centrifugation, 1 mL of dimethyl sulfoxide was added, and it was gently shaken on a shaker for 5 min. After dissolving 100 μL of dimethyl sulfoxide, the solution was transferred to a 96-well plate, and dimethyl sulfoxide (100 μL) was set as a blank group, and the blank group, air control group and exposure group were measured with a microplate reader (490 nm). absorbance.

(4) Results and Analysis

From the absorbance value, the cell viability was calculated according to the following formula.

Cell survival rate (%)=(absorbance of exposure group-absorbance of blank group)/(absorbance of air control group-absorbance of blank group)*100%.

The cytotoxicity of HNB cigarette smoke obtained is shown in Table 1.

Table 1 Cytotoxicity of heat-not-burn cigarette smoke

Example 2 In vitro toxicity evaluation of cigarette smoke labeled with 1 mg tar

The operation steps of the present embodiment are the same as those of the first embodiment. As the exposure dose increases, the cell death rate increases accordingly. The cytotoxicity of flue gas is shown in Table 2.

Table 2 Cytotoxicity of 1mg cigarette smoke

Example 3 In vitro toxicity evaluation of 3R4F cigarette smoke

The operation steps of the present embodiment are the same as those of the first embodiment. As the exposure dose increases, the cell death rate increases accordingly. The cytotoxicity of smoke is shown in Table 3.

Table 3 Cytotoxicity of 3R4F cigarette smoke

It can be seen from Tables 1-3 that there is little difference in the cell viability of the three cigarettes at low smoke exposure doses (10% and 20%). The resulting cell viability had a very significant difference (P<0.01), and the cell viability was heat-not-burn>1mg>3R4F.

Although the present invention has been described in detail above with general description, specific embodiments and tests, some modifications or improvements can be made on the basis of the present invention, which is obvious to those skilled in the art . Therefore, these modifications or improvements made without departing from the spirit of the present invention fall within the scope of the claimed protection of the present invention.

Claims (10)

1, A method for determining cigarette smoke cytotoxicity under gas-liquid interface exposure, which is characterized in that the gas-liquid interface exposure method and the thiazole blue method are combined to detect the cytotoxicity caused by the cigarette smoke,

in the gas-liquid interface exposure method, the number of cells is 0.8 to 1.2 x 10⁶The density of each hole is inoculated on the plug-in culture plate; and (3) carrying out contamination treatment on the cells by using smoke with the flow rate of 8-12 mL/min.

2. The method of claim 1, wherein the distance between the smoke outlet and the permeable membrane of the inserted culture plate is 1.4-1.6 mm during the contamination treatment.

3. The method of claim 1 or 2, wherein the pumping pattern is an ISO 3308:2012 pumping pattern or a canadian deep pumping pattern; preferably ISO 3308:2012 suction mode;

preferably, when the suction mode is ISO 3308:2012 suction mode, the exposure time is 0.5-1.5 h; more preferably 1 h.

4. The method of , wherein the cell is mouse monocyte macrophage RAW 264.7.

5. The method of , wherein the plug-in plate is a 24mm plug-in plate.

6. The method according to of any one of claims 1 to 5, wherein 1.8 to 2.2mL of growth medium is added to the lower chamber and 1.4 to 1.6mL of growth medium is added to the upper chamber during cell culture;

preferably, the cells are infected after 24h of culture.

7. The method according to of claims 1 to 6, wherein the concentration of MTT solution is 0.45 to 0.55mg/mL, preferably 0.5mg/mL, when the thiazole blue method is performed;

preferably, the specific operation of the thiazole blue method comprises the following steps: adding 0.8-1.2 mL of 0.45-0.55 mg/mL MTT solution into the upper chamber of the plug-in culture plate, and adding 5% CO₂Incubating for 2.5-3.5 h at 37 ℃;

and after the preferable incubation is finished, removing the MTT solution in the upper chamber of the plug-in culture plate, adding 0.8-1.2 mL of dimethyl sulfoxide, and slightly shaking for 4-6 min.

8. The method according to any of claims 1-7, wherein each test is performed by providing a clean air exposure chamber into which clean air is introduced as an air control group, providing a smoke exposure chamber into which smoke is introduced as an exposure group, and providing dimethyl sulfoxide as a blank group during absorbance detection;

cell viability ═ 100% (exposure absorbance-blank absorbance)/(air control absorbance-blank absorbance).

9. The method according to claims 1 to 8, comprising the steps of:

(1) cell inoculation: selecting mouse mononuclear macrophage RAW264.7 in logarithmic growth phase at 1 x 10⁶Inoculating the seeds/hole density to a 24mm plug-in culture plate, wherein 1.8-2.2 mL of growth culture solution is added into a lower chamber, 1.4-1.6 mL of growth culture solution is added into an upper chamber, and culturing is carried out for 24 h;

(2) cigarette smoke contamination: after the cells are cultured for 24 hours, the inserted culture plate is moved to an exposure chamber, wherein the upper layer of a microporous membrane of the Transwell chamber is a smoke environment, the lower layer of the microporous membrane of the Transwell chamber is an exposure culture solution, and the cells are positioned at a gas-liquid junction;

setting the distance between a smoke outlet in the exposure chamber and the penetrating membrane of the plug-in culture plate to be 1.4-1.6 mm, and setting the gas flow rate to be 8-12 mL/min;

selecting a pumping mode as an ISO 3308:2012 pumping mode, wherein the exposure time is 0.5-1.5 h;

setting the smoke exposure dose without applying dilution air as 100%, and sequentially diluting smoke to obtain different smoke exposure doses by adjusting the flow rate of dilution air;

each test is provided with a clean air exposure chamber, and clean air is introduced to be used as an air control group; arranging a smoke exposure chamber, and introducing smoke to serve as an exposure group;

(3) MTT cytotoxicity assay: after the flue gas exposure is finished, taking the upper chamber of the inserted culture plate out of the exposure unit, adding 0.8-1.2 mL of 0.45-0.55 mg/mL MTT solution, and adding 5% CO₂Incubating for 2.5-3.5 h at 37 ℃;

centrifuging to remove the redundant MTT solution in the upper chamber, adding 0.8-1.2 mL of dimethyl sulfoxide, and lightly oscillating for 4-6 min;

setting dimethyl sulfoxide as a blank group, and respectively measuring the absorbance of the blank group, the air control group and the exposed group at 490nm by using an enzyme-labeling instrument;

(4) results and analysis: the cell survival rate indicates cytotoxicity, the higher the cell survival rate, the lower the cytotoxicity of the smoke is, otherwise, the higher the cytotoxicity of the smoke is;

cell viability ═ 100% (exposure absorbance-blank absorbance)/(air control absorbance-blank absorbance).

10. Use of the method of any of claims 1-9 in a conventional cigarette or heated non-burning cigarette smoke cytotoxicity test.