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Method for measuring cytotoxicity of condensate of main stream smoke of cigarettes

A technology for mainstream smoke and cytotoxicity of cigarettes, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, and measurement of color/spectral characteristics, etc. Problems such as underestimation of toxic effects

Active Publication Date: 2011-11-09
ZHENGZHOU TOBACCO RES INST OF CNTC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The principle is that serum can be combined with the compound to reduce or mask the toxicity of the compound, so a high concentration of serum will cause the toxic effect of the smoke condensate to be underestimated, making the test results inaccurate; at the same time, complete serum deprivation will affect the normal function of cells to grow

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  • Method for measuring cytotoxicity of condensate of main stream smoke of cigarettes
  • Method for measuring cytotoxicity of condensate of main stream smoke of cigarettes
  • Method for measuring cytotoxicity of condensate of main stream smoke of cigarettes

Examples

Experimental program
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example 1

[0039] Example 1 Evaluation of a certain domestic cigarette

[0040] Use DMSO to extract the smoke particulate phase components captured on the Cambridge filter, and prepare a smoke condensate sample storage solution with a total smoke particulate matter concentration of 10mg / ml, which can be stored at -80°C.

[0041] After digestion of the expanded and cultured A549 cells, the cell suspension (1×10 5 pieces / ml). Add 100 μl of growth medium to each well of the 8 wells in the first column of the 96-well plate as a blank control, add 100 μl of cell suspension to each well, and store at 37°C, 5% CO 2 Conditioned for 24h. The sample dilution culture solution was used to adjust the smoke condensate sample storage solution to different concentrations, and the final concentration of cigarette CSC was set to eight different concentrations of 10, 50, 75, 100, 120, 140, 160, and 200 μg / ml. After the cells were cultured for 24 hours, the culture medium was sucked off, and a group of 8...

example 2

[0051] Example 2 Evaluate a certain domestic cigarette

[0052] Use DMSO to extract the smoke particulate phase components captured on the Cambridge filter, and prepare a smoke condensate sample storage solution with a total smoke particulate matter concentration of 10mg / ml, which can be stored at -80°C.

[0053] After digestion of the expanded and cultured A549 cells, the cell suspension (1×10 5 pieces / ml). Add 100 μl of growth medium to each well of the 8 wells in the first column of the 96-well plate as a blank control, add 100 μl of cell suspension to each well, and store at 37°C, 5% CO 2 Conditioned for 24h. The sample dilution culture solution was used to adjust the smoke condensate sample storage solution to different concentrations, and the final concentration of cigarette CSC was set to eight different concentrations of 10, 50, 75, 100, 120, 140, 160, and 200 μg / ml. After the cells were cultured for 24 hours, the culture medium was sucked off, and a group of 8 well...

example 3

[0063] Example 3 Evaluate a certain domestic cigarette

[0064] Use DMSO to extract the smoke particulate phase components captured on the Cambridge filter, and prepare a smoke condensate sample storage solution with a total smoke particulate matter concentration of 10mg / ml, which can be stored at -80°C.

[0065] After digestion of the expanded and cultured A549 cells, the cell suspension (1×10 5 pieces / ml). Add 100 μl of growth medium to each well of the 8 wells in the first column of the 96-well plate as a blank control, add 100 μl of cell suspension to each well, and store at 37°C, 5% CO 2 Conditioned for 24h. Use the sample to dilute the culture solution to adjust the smoke condensate sample storage solution to different concentrations. The final concentration of cigarette CSC is set to eight different concentrations of 10, 50, 75, 100, 120, 140, 160, and 200 μg / ml. After the cells were cultured for 24 hours, the culture medium was sucked off, and a group of 8 wells in ...

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Abstract

The invention provides a method for measuring cytotoxicity of condensate of main stream smoke of cigarettes, which is characterized by comprising the following steps: 1) preparation of laboratory reagent, 2) preparation of the condensate of smoke, 3) cells inoculation and culture, 4) contamination of the condensate of smoke, 5) neutral red dying, and 6) result and analysis. The content of the method is as follows: on the basis of the principle of neutral red cell toxicity tests, test procedures are improved according to the cytotoxicity characteristics of the condensate of the main stream smoke of cigarettes, and a method for measuring cytotoxicity of condensate of main stream smoke of cigarettes with low hazard is established. In the invention, the step for fixing formaldehyde fixing solution is reduced to cause the test procedures to be simpler, and simultaneously, the condensate of the main stream smoke of cigarettes is a complex mixture system, has the toxic characteristics of lowtoxicity and the like and improves the diluted culture solution of samples, namely that the concentration of fetal calf serum is regulated from 10% to 5%, therefore, the test result is more accurate and reliable. The method has the advantages of simpler operation, higher sensitivity and more reliable results.

Description

technical field [0001] The invention relates to the research field of in vitro toxicological evaluation of cigarette mainstream smoke, in particular to a method for measuring cytotoxicity of cigarette mainstream smoke condensate. Background technique [0002] The in vitro toxicological study of cigarette smoke has become one of the important means to evaluate the health hazards of smoking. At present, most of the in vitro toxicological studies on cigarette smoke focus on the cytotoxicity and genotoxicity of smoke condensate. How to scientifically and accurately evaluate the harm of low-harm cigarette products and tobacco additives, there is no uniform method and procedure in the world. The neutral red test is a widely accepted in vitro cytotoxicity test method used to evaluate the toxicity of drugs, cosmetics, industrial chemical raw materials and environmental pollutants to different cells. Among many cytotoxicity test methods, the neutral red test is also used to evaluate...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/02G01N21/31
Inventor 李翔尚平平聂聪彭斌赵乐刘惠民谢剑平
Owner ZHENGZHOU TOBACCO RES INST OF CNTC
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