The screening of airborne particles concerning the acute pulmonary cytotoxicity by exposing cultivated human lung cells at the ALI using this in vitro exposure system can help to reduce animal experiments. The main advantage of this exposure system is the radial aerosol distribution concept leading to a homogeneous distribution and the position of the particles onto the cells. Before seeding the cells, add 2.5 milliliters of tempered growth medium to each well of a six well plate.
Place the cell culture inserts without cells carefully inside the wells and add one milliliter of growth medium to every cell culture insert. Incubate the plates for 30 minutes at 37 degrees Celsius and 5%carbon dioxide. After trypsinization of cells, dilute 100 microliters of the cell culture suspension in a cup filled with 10 milliliters of isotonic solution.
Tilt the cup slowly without shaking. On a cell counter, determine the number of viable cells per milliliter and cell viability based on the cell specific measurement parameters of A549 cells. After tempering of the plate, aspirate the medium within the cell culture inserts and seed one milliliter of A549 cells with a density of three times 10 to the fifth cells per milliliters in each cell culture insert.
Distribute the cell suspension by gentle rocking and incubate for 24 hours. Set the pressing time via the time control on the front side of the press. Open the compressed air supply at the compressed air valve.
Using the pressure regulator on the front side of the press, set the compressed air pressure to approximately two bar. Pull out the drawer, press the Press button, and read the pressing pressure on the digital pressure switch. Fill the substance container with a small amount of the test substance.
Insert the plunger into the substance container and turn it slightly back and forth to evenly distribute the powder in the container. Place the substance container, with the plunger, in the drawer and press the Press button. Open the drawer and remove the plunger.
A critical step is the pressing of test substances that have to be specifically compressed to a powder cake within the substance container in order to enable stable particulate exposures. Remove the inlet adaptor and the condensate reflector from the aerosol guiding module. Close the three aerosol feeding bores in the aerosol guiding module with plugs and the medium supply connections at the sampling module with dummy flaps.
Connect the vacuum lines with the tube connector of the aerosol guiding module. Use the hand wheel to close the module and measure the value of the flow controllers. A couple minutes after closing, the values decrease below five milliliters per minute.
Start the aerosol generator software. If the substance scraper is not in the lowest position, press the button Homing Mode. Place the substance container with the pressed test material upside down over the substance scraper.
Ensure that the glass of the substance container faces the front. Place the locking plate in the slot over the substance container and tighten the black screw. Change the values for Feed and Rotation to the desired settings.
Use the downward arrows to push down the slide with the substance container until the substance scraper is near the pressed substance. Open the compressed air supply to the aerosol generator with a tap of the mass flow controller and start the aerosol generation by clicking on the Start button. Set the feed rate to 15 to 20 millimeters per hour to avoid long waiting times.
Control the correct particle generation by observing the fine dust cloud with a small flashlight positioned from below behind the glass tube of the elutriator. Start the medium supply with preheated exposure medium and fill the sampling modules until the downpipes are covered while the module is open. Insert blind cell culture inserts into the exposure module, pump the exposure medium down until the downpipes are covered with medium and the lower side of the inserts are in contact with medium.
Start the aerosol generator, close the exposure module, and connect the exposure module to the exposure module outlet of the aerosol generator. 30 minutes after the lead time, seal the exposure module outlet of the elutriator with a rubber plug and remove the blind inserts. Refill the exposure medium until the downpipes are covered with medium.
Remove the cell culture inserts from the six well plates with the help of tweezers. Pour the growth medium carefully from the cell culture inserts off by toppling the inserts and use a pipette to aspirate and discard the residual liquid. Place the inserts in the exposure chambers of both the exposure and clean air modules.
Close the modules and start the exposure experiments by connecting the exposure module to the exposure module outlet of the aerosol generator and the clean air module to the carrier gas supply simultaneously. After completion of the experiment, disconnect the exposure and clean air modules and seal the exposure module outlet. Stop the compressed air supply and the aerosol generator by clicking on the Stop button.
Open the exposure and clean air module and use tweezers to transfer the cell culture inserts to the prepared post-incubation six well plates. Incubate the six well plates, as well as the unexposed cell culture inserts, for 24 hours at 37 degrees Celsius and 5%carbon dioxide at the air liquid interface. 24 hours after exposure, insert the cell culture inserts in the new prepared six well plates.
Add one milliliter of the fresh prepared WST1 solution to each cell culture insert. Rock the plates carefully in order to distribute the solution homogeneously on the cells. Incubate the six well plates with the cell culture inserts for one hour at 37 degrees Celsius and 5%carbon dioxide.
After that, transfer 100 microliters of the supernatant in triplicates from each six well plate to a 96 well plate. Using a microplate reader, measure the absorbance at 450 nanometers with a reference wavelength of 650 nanometers. The CULTEX RFS is a specially designed modular in vitro exposure system that enables the direct and homogenous exposure of cells to airborne particles at the air liquid interface.
Exposure of cells to clean air lead to no decrease of cell viability over time. The exposure system was successfully validated and established as a prediction model for acute inhalation hazards of the tested compounds. Exposure of A549 cells to different test substances that exhibited no, medium, or strong toxicity.
Please ensure that you strictly keep the time frames that are the incubation times, the lead time of particulate generation, and the particulate deposition times. This exposure method is primarily designed for the particulate exposure of cells, but can be extended to the exposure of liquid aerosols and gasses by adapting the aerosol generation method. This screening method enables the qualitative assessment of inhalable particles regarding the acute inhalation toxicity in vitro, thereby reducing animal testing that would normally provide this toxicological assessment.
Don't forget that working with toxic test substances can be extremely hazardous and precautions, such as wearing gloves, a mask, should always be taken when handling such compounds.
