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Multi-channel gas-liquid interface cell direct exposure exposure technology - CultexRFS Compact
Multi-channel gas-liquid interface cells directly exposed to the poisoning technology - CultexRFS Compact, direct exposure of the cell's gas-liquid interface. In the past few decades, air pollution has shown phenomenal speeds, especially the smog pollution in China this year. A sharp increase in the concentration of particulate matter in the air causes an acute or chronic disease of the respiratory tract. In traditional research, the inhalation exposure test of animals is used to determine the health effects of inhalable substances. So far, there are few research methods for exploring alternative animal experiments.
An experimental method for direct contact of air with cells at the gas-liquid interface was proposed by Professor Aufderheide of Cultex. The top surface of the cell is directly exposed to a constant flow atmosphere with a medium supply at the bottom and a constant temperature and CO 2 environment. In 2009, Cultex company presents classical radiation airway flow design (Radial Flow System -Cultex ® RFS) , Cultex RFS cells in vitro method known as the most successful technology exposure.
In 2014, Cultex introduced the Cultex RFS Compact, a third-generation cell exposure system, with direct exposure to the cell's gas-liquid interface. Based on the RFS technology, compact increases the number of cell culture exposure chambers, a total of 6 cell culture inserts, and is divided into two groups. One group (three cell culture inserts) can be exposed to haze particles, and the other group. Conduct a comparative experiment with clean air exposure, or all of the 6 chambers for exposure to the test substance/clean air.
The 6 chambers in the system are radially distributed to ensure uniform distribution of particulate matter to each chamber. The electric heating system provides optimal temperature conditions for in vitro culture of cells, and a dedicated CO 2 access unit ensures longer in vitro exposure of cells. The system is integrated into a bracket with a locking device to ensure the airtightness of the system. Cultex RFS Compact was tested by dose-response of aerosols of different test substances in Cultex laboratory, and dose-dependent of cytotoxicity of test substances at different exposure times. High experimental repeatability and reliability.
Materials and Method
1, Cultex RFS Compact
2. 6.5mm or 12mm cell culture inserts
3, Bronkhorst high precision MFC
4, medium perfusion system
5. Cell line (ATCC: ccl-185) and isolated primary normal human bronchial epithelial cells (NHBE)
Figure 1: CULTEX® RFS Compact for two gas path distributions
Left: Three cell culture inserts are exposed to the test aerosol, and the other three are exposed to clean air in parallel.
Right: Six cell culture inserts are all exposed to the test aerosol or clean air
Particulate exposure experiments were performed in Cultex® RFS Compact, cells were seeded in a well cell culture insert (COSTAR®/Corning) and exposed to direct exposure to particulate matter at the gas-liquid interface for 24 hours. During the experiment, the bottom of the cell was continuously supplied with medium and the environment was stabilized at 37 ° C by an electric heating system.
Cigarette smoke (all flue gas/gas phase-K3r4F) and 1.6% nicotine solution (electron cigarette) were used as test substances for cell exposure experiments.
result
Cell viability was determined by the WST-1 method. For cellular exposure to e-cigarettes, the level of cellular oxidative stress was simultaneously analyzed. Results analysis was performed after exposure 24. The results were compared to the clean air control group (Figures 2 and 3). There are significant differences between the two groups, and the symbols (asterisks) are defined as follows:
**** p < 0.0001 is very significant, *** p = 0.0001 – 0.001 is very significant,
** p = 0.001 – 0.01 is significant, * p = 0.01 – 0.05 significant
Figure 2: Analysis of the cytotoxic effects of cigarette smoke (full flue gas/gas phase) on A549 cells using the WST-1 assay. The results were compared to the clean air control group. Data are given as means + SD from 3 independent experiments with 3 samples each.
Compared to clean air, 10 cigarette mainstream smoke reduces the activity of A549 cells. With the exposure of more flue gas, the cell activity is gradually reduced, and the effect of total smoke on cell viability is more obvious than that of smoke. There was no significant change in the activity of cells exposed to clean air and cells in the cell incubator.
Figure 3: Cytotoxicity and oxidative stress response of cigarette smoke and 1.6% nicotine solution (e-cigarette) after exposure to NHBE. The results were compared to the clean air control group. Data are given as means + SD from 3 independent experiments with 3 samples each.
The effect of cell viability and oxidative stress levels on NHBE cells is relative. Cells exposed to e-cigarette fluid have reduced cellular activity and increased levels of oxidative stress. Exposure to mainstream smoke, lower cell survival and higher oxidative stress. Cells in the clean air control group exhibited higher oxidative stress than cells cultured in the incubator as a result of the mechanical shear force stimulation of the clean air stream. Therefore, the difference in cell viability between the incubator and the clean air control is not significant.
Conclusion and Outlook
• CULTEX® RFS Compact is suitable for direct exposure of six 6.5 mm or 12 mm existing universal cell culture chambers (COSTAR® and Falcon®).
• Two parallel experiments can be performed in each CULTEX® RFS Compact, three cell culture inserts for the test object and three parallel for clean air or different concentrations of test substance.
• Data show good dose effects and high experimental repeatability.
• Suitable for various test substances such as cigarette smoke, nanoparticles, PM2.5, PM10, VOCs, smog mixtures, dry powder particles and atomized aerosols.