In vitro Cannabis Exposures of Lung Epithelial Cells at the Air-Liquid Interface

Our lab studies inhalation toxicology using preclinical models to investigate the health effects of tobacco, e-cigarettes, and cannabis. Focusing on respiratory disease and developing human relevant exposure systems for translational research. Commercialization of advanced exposure systems enables use of ALI models, improving our ability to mimic human lung physiology and study epithelial responses to complex inhaled mixtures like smoke and vapor.

A key challenge is the lack of standardized methods to measure and import compound deposition in ALI models. And this makes it difficult to compare results across studies that use advanced exposure systems. Our protocol addresses the lack of standardized guidelines for ALI and advanced exposure systems, and this helps researchers to improve the reproducibility and consistency in inhalation toxicology studies. Our protocol offers real-time dosimetry using the QCMs, which allow for precise measurement of cannabis product deposition during exposures. This improves the accuracy and reproducibility compared to techniques that lack direct dose monitoring.

[Instructor] To begin, turn on the inExpose exposure system. Choose the experiment template. Input the operator field and click Okay. Switch on the temperature controls to 35 degrees Celsius for the bottom half and 50 degrees Celsius for the top half. After 20 minutes, perform the Microflow leak test using the Microflow test kit. To do so, connect the four ends of the tubing to the microflow tubes. Then, connect the other end to the top of a rotameter. Next, plug input port. Add flow test inserts to expoCube and connect rotameter to the air output of the expoCube. To test the pump flow, reattach the isokinetic sampler to the expoCube. Cap the nebulizer lid and attach bottom of the rotameter to the expoCube output. Next, grind the cannabis flower using an herb grinder to prepare for vaporization. Weigh out 0.25 gram aliquots of ground cannabis and organize each portion using a pill separator. Pack the vaporizer oven with 0.25 grams of prepared cannabis. Configure the quartz crystal microbalance or QCM exposure system. Attach each QCM to its electronics module using the clip connector and connect the module to the inExposed inhalation exposure platform using a seven pin connector. Place QCM inserts into a well plate on the exposure side of the system, ensuring proper alignment of the QCMs. Preheat the exposure system with the QCMs attached until the system stabilizes at 35 degrees Celsius and 50 degrees Celsius. Then activate micro flows and exposure flows as per the experimental settings. Next, use the data acquisition software calibration wizard to calibrate each QCM prior to the exposure run. Wait for the blue calibration bar to stabilize, then click Next to record the QCM resonant frequency for accurate mass calculations. Set the vaporizer to smart path level two temperature setting. Now attach the cannabis vaporizer to the ends unit of the exposure system with a secure gasket. Start the airneb task to increase relative humidity in the system to greater than 75% before introducing the cells. Select a puff profile using the flexi wear data acquisition software. Then record the microflow pressure of 80 kilo Pascals. Top assembly temperature of 50 degrees Celsius, bottom assembly temperature of 35 degrees Celsius, primary exposure flow of 0.3 liters per minute, and a primary control flow of 0.3 liters per minute. Monitor the QCM output signals throughout the exposure period using the connected software interface. To maintain accuracy, clean the QCM after each experiment. Add one milliliter of 70% ethanol inside each QCM insert. Pipet the alcohol up and down two to three times to dissolve any vapor residue. Remove the alcohol solution and allow the QCM to air dry completely in a dust-free environment before the next use. Place the QCM insert into a well plate on the exposure side of the expoCube, ensuring proper alignment. Then open the exposure system and place the cell culture containing respiratory cells played inside the system. Select a puff profile to begin the puff regimen for the desired duration. Enter the control panel settings in the popup window. When using the QCM, monitor real-time tracking of vapor deposition during the exposure. Post-exposure, return the cell culture plate to the incubator until the scheduled assay time point. Collect samples as needed from various compartments including the basal lateral compartment, the apical wash for surfactant or mucus, and the cell lysate. The exposure flow humidity was successfully maintained between 65% and 85% relative humidity over a period of 700 seconds, showing consistent cyclical fluctuations. Delta nine tetrahydrocannabinol deposition in exposed wells, showed consistent delivery with minor variation between wells. QCM analysis confirmed a steady increase in deposited mass in all four wells over 30 minutes, indicating continuous particle delivery throughout the exposure period.