In Vitro Method to Control Concentrations of Halogenated Gases in Cultured Alveolar Epithelial Cells

This method can help answer key question in the research field of lung injury and repair such as testing halogenated adjunct as potential novel therapies for acute respiratory distress syndrome. The main advantage of this technique is that it relies on a very simple method to culture the cells and to expose them to halogenated agents in order to reach precise and controlled concentrations. To construct the airtight chamber, use a hermetic polypropylene box with a capacity of 6.5 liters.

Drill a 2.5 centimeter diameter hole on the bottom side of the lateral wall of the box. Then insert a corrugated tube with a green mark which will serve as the gas air mixture input pipe. And seal it with silicone.

Next, drill a second hole with a 2.5 centimeter diameter on the top side of the opposite lateral wall. Insert another corrugated tube with a red mark and connect it with a charcoal filter which will serve as the gas air mixture output pipe. Then seal it with silicone.

Subsequently drill a tight hole with a four millimeter diameter at the center of the wall. Insert the short infusion tubing that is connected at a manifold with a rotating male lure lock. Plug it in to a gas analyzer and seal it with silicone.

Then place a digital thermometer inside the airtight chamber. Under a laboratory extractor hood, customize an anesthetic machine circuit to switch the nitrous oxide gas line by carbon dioxide. Insert a heated humidifier into the pipe between the anesthetic machine and the airtight chamber to warm the gas flow mixture to approximately 37 degrees Celsius.

Plug the airtight chamber with the green marked corrugated tube into the customized anesthetic machine circuit. Next, place the airtight chamber on a hot plate set to 37 degrees Celsius. Place a six well plate containing human alveolar epithelial cells into the airtight chamber and seal the lid.

Regulate the gas flow rate to quickly obtain 5%of CO2. Then open the halogenated agent evaporator and adjust the desired percentage. Once the target values are achieved, reduce the fresh gas flow rate to one liter per minute.

The airtight chamber can be maintained with this gas flow rate as long as necessary for the experiment. The courses of the sevoflurane and isoflurane concentrations in the medium were similar over time. Immediately after the required concentration of halogenated agent was set, concentrations rose over the first hour.

A plateau was then reached which persisted until the administration of the halogenated agent was stopped. After administration interruption, concentrations decreased within one hour. Shown here are the fractions of halogenated agent over time in the airtight chamber measured by the gas analyzer.

This procedure is relatively inexpensive and very easy to adopt even when researchers have never manipulated an airtight chamber before. Following this procedure, other methods like protein and RNA quantification, immunofluorescence, measurement of protein permeability and free transport can be performed in order to answer questions on the specific effects of the halogenated agents during epithelial lung injury and its resolution. After its development, this technique may pave the way for research in the field of acute respiratory distress syndrome to further study the mechanism involved in epithelial lung injury during ARDS and to test novel therapies.