Flue gas condensate cytotoxicity determination method based on cell electronic sensor

Abstract

A flue gas condensate cytotoxicity determination method based on a cell electronic sensor is characterized by comprising the following technology steps:1) preparation of experiment reagents; 2) preparation of a flue gas condensate; 3) cell inoculation culture; 4) flue gas condensate contamination; 5) results and analysis. The invention utilizes a cell sensor chip to monitor cell growth conditionsand influences of different condensate concentrations on cell growth after contamination of cells by a cigarette mainstream flue gas condensate on a real time basis, so as to establish a new, real-time, mark-free and high-flux method for cigarette mainstream flue gas cytotoxicity determination. Compared with a neutral red absorption method, the method of the invention omits a neutral red dyeing step and a complicated cleaning step to simplify a test process; meanwhile, quality control on cells is carried out according to a real-time cell growth curve after inoculation to optimize contamination time and contamination concentration, and monitor influences of different condensate concentrations on the cell growth curve after contamination of cells on a real time basis, so as to obtain a moreaccurate and reliable experiment result.

Description

technical field

[0001] The invention relates to a technique for measuring the cytotoxicity of cigarette mainstream smoke condensate, in particular to a new method for measuring the cytotoxicity of smoke condensate based on a real-time, label-free, high-throughput cell sensor chip. Background technique

[0002] In vitro toxicology tests are of great significance for human health risk assessment, cigarette hazard assessment and toxicity determination of cigarette additive materials. Among them, the neutral red test, one of the methods recommended by CORESTA, is a widely accepted and feasible in vitro cytotoxicity test method. However, the neutral red test adopts the endpoint detection method, and the selection of the detection time point has a significant impact on the detection results. The influence of the neutral red dye itself on the state of the cells cannot be eliminated. In addition, neutral red dye staining, multiple washing steps increase the possibility of introduc...

Examples

example 1

[0044] 1. Reagents and instruments:

[0045] Dimethyl sulfoxide DMSO (biological reagent pure, purity higher than 99%), MTT dye. CHO-K1 Chinese hamster ovary cell substrain was purchased from the Cell Resource Center of Shanghai Institute of Biological Sciences, Chinese Academy of Sciences. The specific components of the CHO-K1 cell culture medium are 2 mM L-glutamine, 1.5 g / L sodium bicarbonate in F12K, 90%; fetal bovine serum, 10%. xCELLigence cell electronic sensor (Roche Applied Science and ACEA Biosciences), 96-well cell electronic sensor chip. AE163 electronic balance (sensitivity: 0.0001 g) (Mettler, Switzerland).

[0046] 2. Preparation of flue gas condensate stock solution

[0047] First, the cigarette sample A was balanced under ISO standard conditions, and the cigarette was smoked according to the FTC smoking mode, and the total particulate matter (TPM) in the mainstream smoke was collected through an electrostatic collection tube. The total particulate matter...

example 2

[0053] As described in Example 1, the IC of the smoke condensate of cigarette sample B and sample C was measured 50 The values ​​were 94.7 μg / mL and 115.9 μg / mL, respectively.

[0054] At the same time, the neutral red absorption method recommended by CORESTA and the classic method MTT method were used to evaluate the cytotoxicity of the smoke condensate of cigarette samples A and B. The test results of the three methods are shown in Table 1. As can be seen from the table, the cell electron The cytotoxicity determination results of the sensor method are consistent with the neutral red method and MTT method, but it has the advantages of rapidity, accuracy, label-free, simple steps and good reproducibility.

technical field

[0001] The invention relates to a technique for measuring the cytotoxicity of cigarette mainstream smoke condensate, in particular to a new method for measuring the cytotoxicity of smoke condensate based on a real-time, label-free, high-throughput cell sensor chip. Background technique

[0002] In vitro toxicology tests are of great significance for human health risk assessment, cigarette hazard assessment and toxicity determination of cigarette additive materials. Among them, the neutral red test, one of the methods recommended by CORESTA, is a widely accepted and feasible in vitro cytotoxicity test method. However, the neutral red test adopts the endpoint detection method, and the selection of the detection time point has a significant impact on the detection results. The influence of the neutral red dye itself on the state of the cells cannot be eliminated. In addition, neutral red dye staining, multiple washing steps increase the possibility of introduc...

Examples

example 1

[0044] 1. Reagents and instruments:

[0045] Dimethyl sulfoxide DMSO (biological reagent pure, purity higher than 99%), MTT dye. CHO-K1 Chinese hamster ovary cell substrain was purchased from the Cell Resource Center of Shanghai Institute of Biological Sciences, Chinese Academy of Sciences. The specific components of the CHO-K1 cell culture medium are 2 mM L-glutamine, 1.5 g / L sodium bicarbonate in F12K, 90%; fetal bovine serum, 10%. xCELLigence cell electronic sensor (Roche Applied Science and ACEA Biosciences), 96-well cell electronic sensor chip. AE163 electronic balance (sensitivity: 0.0001 g) (Mettler, Switzerland).

[0046] 2. Preparation of flue gas condensate stock solution

[0047] First, the cigarette sample A was balanced under ISO standard conditions, and the cigarette was smoked according to the FTC smoking mode, and the total particulate matter (TPM) in the mainstream smoke was collected through an electrostatic collection tube. The total particulate matter...

example 2

[0053] As described in Example 1, the IC of the smoke condensate of cigarette sample B and sample C was measured 50 The values ​​were 94.7 μg / mL and 115.9 μg / mL, respectively.

[0054] At the same time, the neutral red absorption method recommended by CORESTA and the classic method MTT method were used to evaluate the cytotoxicity of the smoke condensate of cigarette samples A and B. The test results of the three methods are shown in Table 1. As can be seen from the table, the cell electron The cytotoxicity determination results of the sensor method are consistent with the neutral red method and MTT method, but it has the advantages of rapidity, accuracy, label-free, simple steps and good reproducibility.

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