The overall goal of this procedure is to generate liquid extracts from electronic cigarette aerosols for use in toxicological assays. This method can help answer important questions about electronic cigarettes, such as how does electronic cigarette aerosol affect human vascular cells? The main advantage of this technique is that it allows for the airflow and puff-timing protocol to be customized by any individual lab to meet their specific research needs.
The implications of this technique extends toward a better understanding of the potential risks of electronic cigarette use, because it provides a customizable platform that can generate electronic cigarette aerosol extracts in your own lab. This method can provide insight into the risks of electronic cigarettes. It can also be applied to other products, such as conventional tobacco products or cannabis products.
To begin, secure a 100-milliliter Buchner flask to a steel ring stand and create a vacuum trap by filling it with 50 grams of calcium chloride to serve as a desiccant. Next, seal the flask with a rubber through-hole stopper. Wrap the stopper junction with paraffin film and run a pipette through the hole.
Using vinyl tubing, connect the pipette extending from the topper to a T-intersection hose connector. Then use it to connect the two impingers to each other and to connect the output of the second impinger to the T-intersection hose connector. Next use the vinyl tubing to connect the input port of the first impinger to serve as an inhalation port.
Also connect the sidearm of the Buchner flask to the input port of an airflow regulator. And connect the exit port of the airflow regulator to a vacuum pump. Then place a 510 threaded base into a ring stand clamp and use vinyl tubing to connect the solenoid valve to the free end of the T-intersection hose connector.
When finished, check that all of the joints are airtight and apply hose clamps and vacuum grease as needed. First, determine the mass of each e-cigarette cartomizer or tank before they are used by placing them on an analytical balance. The weight difference between pre and post-use will determine the appropriate dosing.
For commercial 3R4F research cigarettes, the nicotine content will be 7 milligrams. For all other commercial cigarettes, the nicotine content should be determined by conventional analytical methods. When using the neutral red uptake cell viability assay for downstream analysis, fill the reservoir of the primary impinger with 4.3 milliliters of endothelial cell culture medium.
Next, prep the electronic cigarette or conventional cigarette for extraction. To prepare a conventional cigarette for extraction, apply a piece of clear tape around the filter and put an easily visible mark where the cigarette paper joins the filter. To prepare an e-cigarette, make sure that the battery is well charged and that the cartomizer is tightly screwed to the battery.
If using an electronic cigarette tank, make sure that an appropriate volume of electronic cigarette liquid is loaded in the tank and screw the tank onto the 510 threaded base. For the conventional cigarette, load it into the inhalation port and secure with a hose clamp. Then turn on the vacuum pump.
Adjust the flow meter to pull 1.65 liters per minute to ensure a 55-milliliter puff over two seconds. Then turn on the microcontroller and use it to light the cigarette on the first puff. Run the pump until the projected desired concentration is achieved.
For an e-cigarette, start by loading it into the inhalation port and securing it with a hose clamp. As with the conventional cigarette, turn on the vacuum pump and adjust the flow meter to pull 1.65 liters per minute to ensure a 55-milliliter puff over two seconds. Run the pump until the projected desired volume has been used.
Then stop the vacuum and determine the post-use mass of each e-cigarette cartomizer or tank using an analytical balance. Compare this to the pre-use mass in order to determine the total mass consumed. If an insufficient mass was consumed, return the electronic cigarette to the device and run the extraction for additional time.
After each extraction, rinse the tubing and reservoirs of the device with 70%ethanol and deionized water to prevent carryover between samples. Then briefly run the empty device to allow airflow to assist with drying of the lines. If the extract is to be used for cell culture, filter the extract containing media through a 0.22-micron polyethersulfone syringe filter.
One day prior to running the assay, seed human umbilical vein endothelial cells into 96-well plates at 10, 000 cells per well in 100 microliters of endothelial cell growth medium. Incubate the cells for 24 hours at 37 degrees Celsius and 5%CO2. The next day, replace the old culture medium with either 100 microliters of fresh endothelial cell culture medium to serve as a control or 100 microliters of endothelial cell growth medium containing 500 micromoles of consumed nicotine to serve as the treatment group.
Seal the plate with a foil seal to keep the wells airtight, and then place the plate back into the incubator for 18 to 24 hours. Many of the components of cigarette smoke and electronic cigarette aerosol are volatile, so it is important to cover the wells with a foil seal so that the wells will be airtight. Shortly before its use, dilute a 100x neutral red stock solution 1 to 100 in cell culture medium to create a 1x neutral red staining solution.
Incubate the neutral red staining solution at 37 degrees Celsius for at least 30 minutes prior to its use. It's normal for some crystals to precipitate during incubation. To avoid applying these crystals to the cell culture wells, pass the solution through a 22-micron filter.
Remove the experimental culture medium and add 100 microliters of the neutral red staining solution to each well. Use the excess stain to create at least three blank wells for proper quantification. Incubate the plate for two to four hours, then remove neutral red staining solution and wash the cells three times by submersion in PBS.
Next, apply a neutral red de-stain solution and incubate the plate for at least ten minutes at room temperature while shaking. When finished, read the plate at an absorbance of 540 nanometers. Within 24 hours of the exposure of human umbilical vein endothelial cells to either conventional cigarette smoke extract or electronic cigarette aerosol extract, there is a significant reduction in cell viability.
In addition to cell viability studies, the extracts themselves can be analyzed using gas chromatography mass spectrometry if extracted into acetone. Using this method, many components of the extract can be determined. Don't forget that working with aerosol or smoke can be hazardous and that all extractions should be performed in a chemical safety hood.
