CN110736696A - method for determining cigarette smoke cytotoxicity under exposure of gas-liquid interface - Google Patents
method for determining cigarette smoke cytotoxicity under exposure of gas-liquid interface Download PDFInfo
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- 239000000779 smoke Substances 0.000 title claims abstract description 87
- 235000019504 cigarettes Nutrition 0.000 title claims abstract description 46
- 238000000034 method Methods 0.000 title claims abstract description 45
- 231100000135 cytotoxicity Toxicity 0.000 title claims abstract description 29
- 230000003013 cytotoxicity Effects 0.000 title claims abstract description 29
- 239000007788 liquid Substances 0.000 title claims abstract description 20
- 238000001514 detection method Methods 0.000 claims abstract description 19
- FZWLAAWBMGSTSO-UHFFFAOYSA-N Thiazole Chemical compound C1=CSC=N1 FZWLAAWBMGSTSO-UHFFFAOYSA-N 0.000 claims abstract description 9
- 210000004027 cell Anatomy 0.000 claims description 38
- 239000000243 solution Substances 0.000 claims description 29
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 24
- 238000002835 absorbance Methods 0.000 claims description 16
- 238000005086 pumping Methods 0.000 claims description 10
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- 238000012360 testing method Methods 0.000 claims description 7
- 231100000263 cytotoxicity test Toxicity 0.000 claims description 6
- 238000011081 inoculation Methods 0.000 claims description 6
- 210000002540 macrophage Anatomy 0.000 claims description 6
- 230000003833 cell viability Effects 0.000 claims description 5
- 239000001963 growth medium Substances 0.000 claims description 5
- 239000012528 membrane Substances 0.000 claims description 5
- 238000002372 labelling Methods 0.000 claims description 4
- UGFAIRIUMAVXCW-UHFFFAOYSA-N Carbon monoxide Chemical compound [O+]#[C-] UGFAIRIUMAVXCW-UHFFFAOYSA-N 0.000 claims description 3
- 230000003698 anagen phase Effects 0.000 claims description 3
- 238000004458 analytical method Methods 0.000 claims description 3
- 238000002784 cytotoxicity assay Methods 0.000 claims description 3
- 238000007865 diluting Methods 0.000 claims description 3
- 239000003546 flue gas Substances 0.000 claims description 3
- 239000007789 gas Substances 0.000 claims description 3
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- 238000004113 cell culture Methods 0.000 claims description 2
- 238000012258 culturing Methods 0.000 claims description 2
- 238000011534 incubation Methods 0.000 claims description 2
- 210000001616 monocyte Anatomy 0.000 claims description 2
- 230000035945 sensitivity Effects 0.000 abstract description 7
- 235000019505 tobacco product Nutrition 0.000 abstract description 6
- 241000208125 Nicotiana Species 0.000 abstract description 5
- 235000002637 Nicotiana tabacum Nutrition 0.000 abstract description 5
- 238000011156 evaluation Methods 0.000 abstract description 5
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- 238000010438 heat treatment Methods 0.000 abstract description 2
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- 238000002474 experimental method Methods 0.000 description 4
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- 229910002092 carbon dioxide Inorganic materials 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 2
- 229910000397 disodium phosphate Inorganic materials 0.000 description 2
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- GLNADSQYFUSGOU-GPTZEZBUSA-J Trypan blue Chemical compound [Na+].[Na+].[Na+].[Na+].C1=C(S([O-])(=O)=O)C=C2C=C(S([O-])(=O)=O)C(/N=N/C3=CC=C(C=C3C)C=3C=C(C(=CC=3)\N=N\C=3C(=CC4=CC(=CC(N)=C4C=3O)S([O-])(=O)=O)S([O-])(=O)=O)C)=C(O)C2=C1N GLNADSQYFUSGOU-GPTZEZBUSA-J 0.000 description 1
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- 239000000463 material Substances 0.000 description 1
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- PGSADBUBUOPOJS-UHFFFAOYSA-N neutral red Chemical compound Cl.C1=C(C)C(N)=CC2=NC3=CC(N(C)C)=CC=C3N=C21 PGSADBUBUOPOJS-UHFFFAOYSA-N 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
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- 238000010998 test method Methods 0.000 description 1
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Abstract
The invention relates to the field of detection of tobacco and tobacco products, in particular to methods for determining cigarette smoke cytotoxicity under gas-liquid interface exposure, which detects the cytotoxicity caused by cigarette smoke by combining a gas-liquid interface exposure method and a thiazole blue method (MTT method), wherein in the gas-liquid interface exposure method, cells are exposed at the rate of 0.8-1.2 x 106The detection method can objectively reflect the process that a human respiratory system contacts cigarette smoke, improves the accuracy and sensitivity of detection, reduces the detection time, is more convenient and quicker, can realize the cytotoxicity evaluation of the cigarette smoke after heating and non-burning and the traditional cigarette smoke, and has the advantages of coverage compared with the prior art, simple operation and more comprehensive and reliable result.
Description
Technical Field
The invention relates to the field of detection of tobacco and tobacco products, in particular to methods for determining cigarette smoke cytotoxicity under exposure of a gas-liquid interface.
Background
The cytotoxicity test of cigarette smoke is which is an important means for evaluating the influence of smoking on health, and mainly comprises two steps of detecting the exposure of a tested object and the survival rate of cells after the exposure, wherein the cigarette smoke exposure is step for measuring the cytotoxicity, and the current cigarette smoke exposure mainly adopts two modes of smoke condensate and gas-liquid interface exposure contamination in an actual contact state.
The 2002 international tobacco science research cooperation center recommends that a neutral red test method is used for determining cytotoxicity, but the method has complex operation steps, and errors are easily introduced due to repeated cleaning.
With the increasingly strict tobacco regulatory legislation and deep smoking and health research, tobacco companies strive to find novel tobacco products with lower risks as products for replacing traditional cigarettes in the future, the heated non-combustible cigarettes are used as novel tobacco products, and the release amount of harmful ingredients in smoke is far lower than that of the traditional cigarettes.
At present, no report is found for detecting cytotoxicity caused by cigarette smoke by combining gas-liquid interface exposure and thiazole blue, and in addition, no report is found for detecting the cytotoxicity of a heating non-combustible tobacco product by the combined method.
Disclosure of Invention
In order to solve the technical problems, the invention provides methods for simply and quickly measuring the cytotoxicity of cigarette smoke under the exposure of a gas-liquid interface, which can improve the sensitivity and the accuracy of detection, and the specific technical scheme is as follows:
the gas-liquid interface exposure method and the thiazole blue method (also known as MTT method) are combined to detect the cytotoxicity caused by the cigarette smoke. The method is suitable for the traditional cigarette products and is also suitable for the cytotoxicity detection of the smoke of novel tobacco products.
In the gas-liquid interface exposure method, the number of cells is 0.8 to 1.2 x 106The density of each well is inoculated on an insert culture plate (also called a Transwell culture plate); and (3) carrying out contamination treatment on the cells by using smoke with the flow rate of 8-12 mL/min.
The gas-liquid interface exposure and thiazole blue joint detection method takes fresh smoke as a subject, and can better reflect the process that a human respiratory system contacts cigarette smoke; cells are more sensitive to smoke in the exposure process, so that the exposure time of the test object is shortened; cells do not need to be cleaned in the detection process, so that the loss of the cells is avoided, and the detection accuracy is improved.
However, although the sensitivity of the cells to smoke can be enhanced by the gas-liquid interface exposure method, the cells are easy to die due to non-smoke reasons (the phenomenon that the air control group has more than normal cell death) or the phenomenon that the sensitivity of the cells is reduced (the phenomenon that the number of dead cells in the smoke treatment group is obviously less than the expected condition) occurs, so that the sensitivity and the accuracy of the detection are difficult to be balanced. In contrast, the research of the invention finds that the inoculation density of cells and the flow rate during flue gas treatment have great influence on the problems, and further, through multiple attempts, the optimization is obtained as follows: when the inoculation density of the cells and the flow rate during smoke treatment are both in the limited range, the sensitivity and the accuracy of smoke detection are favorably considered.
Preferably, the cells are inoculated in an amount of 1.0 x 106The flow rate of the smoke is 10 mL/min.
In order to further optimize the detection of smoke, the present invention optimizes other conditions as well, resulting in the following conditions:
preferably, when the contamination treatment is carried out, the distance between the smoke outlet and the penetrating membrane of the inserted culture plate is 1.4-1.6 mm; preferably 1.5 mm.
Preferably, the smoke exposure dose without applying dilution air is set to be 100%, and different smoke exposure doses are obtained by sequentially diluting smoke by adjusting the flow rate of dilution air.
Preferably, the pumping mode is ISO 3308:2012 pumping mode or canadian deep pumping mode; preferably ISO 3308:2012 suction mode.
Preferably, the application also optimizes the smoke exposure dose aiming at the suction mode, and when the suction mode is ISO 3308:2012 suction mode, the exposure time is 0.5-1.5 h; preferably 1 h.
Preferably, the cell is mouse monocyte macrophage RAW 264.7.
Preferably, the plug-in plate is a 24mm plug-in plate.
Preferably, during cell culture, 1.8-2.2 mL (preferably 2mL) of growth culture solution is added into the lower chamber, and 1.4-1.6 mL (preferably 1.5mL) of growth culture solution is added into the upper chamber;
preferably, the cells are infected after 24h of culture.
Preferably, the concentration of the MTT solution is 0.45 to 0.55mg/mL, and preferably 0.5mg/mL, in the case of performing the thiazole blue method.
The invention finds that in the technical scheme of the invention (particularly after the specific cell inoculation number and the specific suction mode are set), the concentration of the MTT solution is set to be in the range, so that the improvement of the detection effect is more facilitated.
Preferably, the specific operation of the thiazole blue method comprises the following steps: adding 0.8-1.2 mL (preferably 1mL) of 0.45-0.55 mg/mL MTT solution in 5% CO in the upper chamber of the plug-in culture plate2Incubating for 2.5-3.5 h (preferably 3h) 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.
Preferably, a clean air exposure chamber is arranged in each test, and clean air is introduced to serve as an air control group; arranging a smoke exposure chamber, and introducing smoke to serve as an exposure group; and setting dimethyl sulfoxide as a blank group during absorbance detection;
cell viability ═ 100% (exposure absorbance-blank absorbance)/(air control absorbance-blank absorbance).
Preferably, each exposure set is set up to consist of three parallel chambers, allowing three parallel experiments per exposure dose point.
The above parallel experiments ensure that the accuracy of the test results is further .
As preferred technical solutions of the invention, the method comprises the following steps:
(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).
Preferably, the growth culture solution and the exposure culture solution are DEME-H basic culture solution and 10% fetal bovine serum.
Preferably, the PBS (0.01M phosphate buffer) is prepared as follows: taking 0.2g of KCl, 0.2g of 0.2gKH2PO4，8gNaCl，2g Na2HPO4·12H20, adding ultrapure water to 1L, measuring pH to 7.2, and autoclaving.
Preferably, the preparation method of the MTT solution is as follows: 0.250g of MTT was weighed, 50ml of PBS was added, dissolved, filtered through a 0.22 μm microporous membrane, and stored at-20 ℃.
Preferably, the smoke is continuously generated and uniformly delivered to the smoke exposure unit using a fully automatic smoking machine using a plurality of smoking modes such as ISO 3308:2012 smoking modes, canadian deep smoking modes, and the like.
The invention further provides the application of the method in the cytotoxicity test of the smoke of the traditional cigarette or the cigarette which is not burnt after being heated.
The invention has the following beneficial effects:
the detection method can more objectively reflect the process that the respiratory system of the human body contacts with the cigarette smoke, not only improves the accuracy and the sensitivity of detection, but also reduces the detection time, and is more convenient and quicker.
Meanwhile, the method can realize the evaluation of the cytotoxicity of the smoke of the heated non-combustible cigarette and the traditional cigarette, has a coverage of more compared with the prior art, and is simple to operate and more comprehensive and reliable in result.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
The reagents and equipment used in the following examples are as follows:
(1) instruments and devices employed
Carbon dioxide incubator (SANYO corporation, japan), enzyme labeling instrument (Bio-tek corporation, usa), gas-liquid interface exposure system (beijing huilong and technology limited, china), full-automatic smoking machine (beijing huilong and technology limited, china), full-automatic cell counter (ThermoFish Scientific, usa), inverted microscope (olpuyms corporation, japan), ultra-clean bench (suzhou altai air technology limited, china), 24mm insertion type 6-well culture plate (Transwell culture plate) (Corning corporation, usa).
(2) Reagents and materials used
Mouse mononuclear macrophage RAW264.7 (ATCC cell bank, usa), DMEM-H basal medium (GENVIEW corporation, germany), Fetal Bovine Serum (Fetal Bovine Serum, FBS) (AusGeneX corporation, australia), 3R4F cigarette (kentucky university reference cigarette, usa), 1mg cigarette (mixed cigarette with 1mg tar in the box, china), heat-not-burn cigarette (original type, purchased from japan), MTT (3- (4, 5-dimethyl-2-thiazoly) -2,5-diphenyl-2-H-tetrazolium bromide, 3- (4, 5-dimethylthiazol-2) -2, 5-diphenyltetrazolium bromide) (purity of 99.0% or more, SIGMA-ALDRICH corporation, usa), trypan blue (purity of 99.0% or more, SIGMA-ALDRICH corporation, usa), dimethyl sulfoxide (purity of 99.0% or more, chemical agents limited of the national drug group, china).
Growth medium (DEME-H basic medium with 10% FBS), exposure medium (DEME-H basic medium with 10% FBS, 0.01M phosphate buffer (0.2g KCl, 0.2 gKH)2PO4，8gNaCl，2g Na2HPO4·12H20, adding ultrapure water to 1L, measuring pH to 7.2, autoclaving), and MTT solution (0.250 g of MTT was weighed, 50ml of PBS was added, and after dissolution, filtration was performed with a 0.22 μm microporous membrane, and split charging was performed at-20 ℃ for storage).
Example 1 in vitro cytotoxicity evaluation of heated non-burning cigarette Smoke
The method specifically comprises the following steps:
(1) cell inoculation and culture
Selecting mouse mononuclear macrophage RAW264.7 in logarithmic growth phase at 1 x 106The density of each well was inoculated into a 24mm plug-in plate (Transwell plate) in which 2mL of growth medium was added to the lower chamber and 1.5mL of growth medium was added to the upper chamber, and cultured for 24 h.
(2) Cigarette smoke contamination
After the cells are cultured for 24h, the Transwell 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 is an exposure culture solution, and the cells are positioned at a gas-liquid junction.
The heated non-combustible cigarettes were smoked according to ISO 3308:2012 smoking pattern (35mL/60s/2s) using a fully automatic smoking machine, and the continuously produced smoke was uniformly delivered to the smoke exposure unit. The distance between the bell mouth in the exposure chamber and the transparent membrane of the Transwell culture plate is 1.5 mm. The gas flow rate of the exposure chamber was set to 10mL/min by a negative pressure pump and a micro flow controller. The smoke exposure without the application of dilution air was set to 100%, and by adjusting the flow rate of dilution air, the smoke exposure was set to 10%, 20%, 40%, 60%, and 80% by adjusting the flow rate of dilution air, and the smoke exposure was set to 1 h. 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; each exposure group consisted of three parallel chambers, allowing three parallel experiments per exposure dose point.
(3) MTT cytotoxicity assay
After the smoke exposure was complete, the upper chamber of the Transwell plate was removed from the exposure unit and 1mL of 0.5mg/mL MTT solution, 5% CO was added2And incubated in an incubator at 37 ℃ for 3 h. Excess MTT solution in the upper chamber was removed by centrifugation, 1mL of dimethyl sulfoxide was added, and the mixture was placed on a shaker and shaken gently for 5 min. After dissolving 100. mu.L of dimethyl sulfoxide, transferring the solution into a 96-well plate, setting the dimethyl sulfoxide (100. mu.L) as a blank group, and measuring the absorbance of the blank group, the air control group and the exposure group by using an enzyme-labeling instrument (490 nm).
(4) Results and analysis
From the absorbance, the cell survival rate was calculated according to the following formula.
Cell viability (%) - (exposure absorbance-blank absorbance)/(air control absorbance-blank absorbance) × 100%.
The cytotoxicity of the obtained smoke of the heated non-combustible cigarette is shown in Table 1.
TABLE 1 cytotoxicity of cigarette smoke without Heat burn
Example 2 in vitro toxicity evaluation of cigarette Smoke labeled with 1mg Tar
The procedure of this example is the same as that of example , with the increase in the amount of the toxicant, the cell death rate increased and the cytotoxicity of the smoke is shown in Table 2.
TABLE 21 cytotoxicity of cigarette Smoke mg
Example 33 in vitro toxicity evaluation of cigarette Smoke R4F
The procedure of this example is the same as that of example , with the increase in the amount of the toxicant, the cell death rate increased and the cytotoxicity of the smoke is shown in Table 3.
TABLE 33R 4F cigarette Smoke cytotoxicity
As can be seen from tables 1-3, the cell survival rates of the three cigarettes are not greatly different at low smoke exposure doses (10% and 20%), and have extremely significant difference (P is less than 0.01) with the increase of exposure doses (40%, 60% and 80%), and the cell survival rates are more than 1mg and more than 3R4F when the cigarettes are not burnt.
Although the invention has been described in detail with respect to , specific embodiments and experiments, it will be apparent to those skilled in the art that variations or improvements may be made therein without departing from the spirit of the 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.
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