This protocol is significant, as the PIVEC allows for direct exposure of a cell culture to to real-world aerosols. The system can be warn to sample the same air a person breathes, enabling a new type of air quality monitoring. The main advantage of this technique is the ease of capturing aerosols on to cultured lung cells at locations inaccessible to bench-top systems, such as at the source of admission or within the breathing zone.
Demonstrating the procedure will be Dr.Lynn Secondo, a recent graduate from my laboratory, and Nathaniel Wygal, an undergraduate student from my laboratory. For this experiment, stored test materials and controlled environment for 24 hours prior to the test. To assemble the dry dispersal system, connect the ball valve to one end of the four inch long, 1/8 size threaded pipe.
This serves as the particle hopper. Through the open end of the particle hopper, put in desired amount of copper nanoparticles. Then close the particle hopper with another ball valve.
Connect the two inch long, 1/8 size pipe to the valve. Place a three inch long, 1/2 inch outer diameter tubing around the two inch pipe. Insert a HEPA filter inside the short tubing, ensuring the flow direction is through the ball valve.
Using threading, connect the vacuum generator to the other ball valve. Then, connect the vacuum generator to the air tank. Put 1/4 inch outer diameter tubing over the outlet of the vacuum generator to connect it to the experimental setup.
Turn the main valve and regulator valves on the air tank to set the desired flow. Open the ball valve, closest to HEPA filter then open the ball valve closest to vacuum generator. Keep them open during characterization or exposure before closing them.
First the ball valve closest to vacuum generator, and then the ball valve closest to HEPA filter. Close main and regulator valves on air tank to stop the flow. To clean up, use 70%ethanol to wash ball valves and vacuum generator.
Put metal pipes in autoclave for sterilization. After culturing cells at air liquid interface, allow cells to equilibrate for 24 hours. To assemble, take the PIVEC base with the well facing up and connect the cell culture insert adapter on the top.
Add four milliliters of cell culture media to the well of the base of the PIVEC using a micropipette. Use tweezers to place the cell culture insert within the adapter. With the narrow part of PIVEC top piece facing up, connect to the adapter.
Carefully, wrap PIVEC with a single layer of duct tape. Push to connect 37 millimeter cassette pieces on top and bottom of PIVEC. Place 1/4 inch barbed adapters into cassette inlet and outlet.
Wrap resistive heater around PIVEC with the wires at the base. Tape to secure. Wrap PIVEC with eight rounds of aluminum foil for insulation, and secure with tape.
Connect 1/2 inch outer diameter tubing to the adapter on the top of PIVEC. Remove porous tubing from sterile water and place within tubing on top of PIVEC. Transfer system to fume hood and clamp the PIVEC on the ring stand and secure.
Connect PIVEC to the aerosol system by connecting the 1/4 inch diameter tubing at the outlet of the aerosol generator to the porous tubing. Connect the exit of the PIVEC to 1/4 inch diameter tubing that is connected to a HEPA filter and a vacuum pump by a Y connector. Turn on the valves and set the pump flow rate to 0.5 liters per minute.
Expose the cells to copper nanoparticles for ten minutes before closing the valves and shutting off the pump. Remove PIVEC from the aerosol system. Take out the cell culture insert using tweezers and place it in the sterile well plate to begin desired biological assays.
Proposed exposure time points, put the insert back to the carbon dioxide incubator. Aspirate media from PIVEC using a dropper. Use 70%ethanol to wash PIVEC and sterilize it with ultraviolet light for at least 30 minutes prior to the next experiment.
The PIVEC was characterized using three sizes of copper nanoparticles to determine deposition efficiency. Increased deposition is observed overall for the 24 well design, then the six well design. It slightly decreases for 100 nanometer in comparison to 40 nanometer and 800 nanometer copper particles.
Particle number concentration of copper nanoparticle aerosols was determined by filter-based scanning mobility particle sizer, as well as optical particle sizer measurements. Post-exposure analysis can be expedited by determining the dose relationship between the 37 millimeter filter and the cell culture insert. The comparison shows a strong correlation for 800 nanometer copper particles with a p-value less than 0.05.
Compared to perpendicular-flow in vitro exposure systems throughout literature, the deposition efficiency of the PIVEC over the range of the particle sizes tested is comparable with, or increased, than reported values. The most important thing to remember is that although this protocol demonstrates the use of the PIVEC in a laboratory setting, it was designed to measure cellular responses to aerosols at the site of exposure. When conducting on-site testing, it is important to characterize the deposition efficiency of the test aerosol.
The deposition efficiency determines the deposited dose of particles to which the cells are exposed. Additional biological assays can be performed to investigate more cellular response endpoints due to exposure. The PIVEC has been used to assess the cellular response due to aerosols generated during 3D-printing processes, such as metal binder jetting, and fused filament fabrication.
Use of nanoparticles, particularly in aerosol form, can be hazardous and any exposures should be performed in a fume hood. Cellular work should be done in a bio-safety cabinet.
