The authors would like to thank Boris Solomonov and the Virginia Commonwealth Innovation Machine Shop for help with rapid prototyping the device. The authors would also like to thank Cristian Romero-Fuentes of the Lewinski Group, Dr. Vitaliy Avrutin, Dr. Dmitry Pestov, and the Virginia Commonwealth Nanomaterials Core Characterization Facility for their help with particle characterization. This work was supported by startup funds provided to Dr. Lewinski by the College of Engineering at Virginia Commonwealth University.

Operators must wear personal protective equipment (e.g. lab coat, gloves, goggles) when performing steps 1, 2, 3, 5, and 6.
1. Preparation of Materials
<ol>
	<li>Prepare materials for system assembly and exposure to ensure repeatability.
	<ol>
		<li>Make sure to use new or 70% ethanol cleaned &#188;&#8221; inner diameter conductive tubing and &#188;&#8221; outer diameter connectors for the system assembly.</li>
		<li>Store test materials including filters, PIVEC components, tweezers, and particle powders in a well-controlled environment, with respect to the temperature and humidity, for at least 24 h prior to the experiment. 
		NOTE: The temperature should be near room temperature, approximately 20 &#176;C, with relative humidity less than 35%. This is very important to achieve repeatability between experiments.</li>
		<li>Prepare particle counters using isopropanol to clean parts and allow for the system warm-up according to the manufacturer recommendations, including the scanning mobility particle sizer (SMPS) and optical particle sizer (OPS) for measurement.</li>
	</ol>
	</li>
</ol>
2. Generation of Dry Aerosol
NOTE: Operators should perform aerosol generation in a fume hood.
<ol>
	<li>Assemble a system to generate dry aerosols 
	NOTE: The suspension of particles in gas or liquid should be appropriate for the modeled application and cell culture. The following method can be performed using a liquid-based aerosol. The design of the dry aerosol system is from Tiwari et al.37 A schematic of the dry dispersal system is shown in Figure 1.
	<ol>
		<li>Connect the ball valve to each end of the 4&#8221; 1/8 size threaded pipe, this will serve as the particle hopper. Connect 2&#8221; 1/8 size pipe to one valve.</li>
		<li>Weigh copper nanoparticles, in this study the mass concentration for each particle size was kept constant while determining the deposition efficiency. Approximately use 7.5 mg of 40 nm copper nanoparticles, 7 mg of 100 nm copper nanoparticles, and 13 mg of 800 nm copper nanoparticles per exposure. Place copper nanoparticles into the particle hopper through the open end. 
		NOTE: The amount of copper nanoparticles weighed will serve as the mass based administered concentration.</li>
		<li>Place a 3&#8221; piece of &#189;&#8221; outer diameter (OD) tubing around the 2&#8221; pipe and place a HEPA filter inside this short tubing such that the flow direction is through the ball valve.</li>
		<li>Connect the vacuum generator to other ball valve using threading. Connect vacuum generator to air tank by placing a 5/16&#8221; OD tubing into the push-to-lock connection. Use &#188;&#8221; OD tubing to connect the outlet of the vacuum generator to the experimental set-up by placing the tubing over the outlet of the vacuum generator.</li>
	</ol>
	</li>
	<li>Use of dry aerosol system to generate dry aerosol
	<ol>
		<li>Open the air tank by turning the main valve and allow the air flow to the system. Open the valve on the flow regulator on the air tank and set such that the flow through the system is equivalent to the desired settings on the vacuum pump.</li>
		<li>Open the ball valve closest to HEPA filter then open the ball valve closest to vacuum generator. Keep these open for approximately 3 s to allow particles to be pulled into the air stream.</li>
		<li>Close the ball valve closest to vacuum generator then close ball valve closest to HEPA filter. Allow air from the tank to flow for the duration of the experiment as necessary.</li>
		<li>Close main and regulator valves on air tank to stop the flow. Clean ball valves and vacuum generator using 70% ethanol. Autoclave metal pipes for sterilization.</li>
	</ol>
	</li>
</ol>
3. Deposition Efficiency Measurement using PIVEC
NOTE: Operators should perform aerosol exposures in a fume hood.
<ol>
	<li>Measure the deposition by collecting the copper nanoparticle aerosol generated in step 2.2 on a pre-weighed filter. Use the deposited dose, measured using the collected mass on the filter, and the administered dose, measured using the amount of weighed copper particles, to determine the deposition efficiency.
	<ol>
		<li>Keep 1.00 &#181;m pore glass fiber filters under low humidity conditions, described in 1.1.2, for at least 24 h prior to pre-exposure measurements. Weigh an unused filter three times and record the filter weights. Place the unused filter in a cell culture insert.</li>
		<li>Choose appropriate cell culture insert adapter (6 well or 24 well) for PIVEC to support the cell culture insert with the filter. Place the cell culture insert adapter piece on the top of the base of PIVEC, setting into place such that the base of the adapter piece is wider than the top.</li>
		<li>Use tweezers to place filter loaded cell culture insert within adapter piece. Place the top piece on top of adapter piece, settling into place such that the base of the top piece is wider than the top. Wrap PIVEC with a single layer of duct tape.</li>
		<li>Connect 37 mm cassette pieces on top and bottom of PIVEC by pushing into place. Place &#188;&#8221; barbed adapters into cassette inlet and outlet.</li>
		<li>Wrap the resistive heater around PIVEC such that the wires are at the base. Tape to secure.</li>
		<li>Wrap PIVEC with ~8 rounds of aluminum foil for insulation. Secure with tape.</li>
		<li>Connect 2&#8221; long piece of 1/2&#8221; outer diameter flexible tubing to the adapter on top of PIVEC. Remove porous tubing from sterile water and place within tubing on top of PIVEC.</li>
		<li>Place PIVEC within clamp on the ring stand and secure. Complete set-up with the vacuum pump, particle counters, and aerosol set-up. 
		NOTE: The number-based deposited dose can be determined only if particle counters are placed before the PIVEC and after the PIVEC on separate runs.</li>
		<li>Expose filters using step 2.2 of protocol and desired exposure time and flow rates, in this study an exposure time of 10 min at 0.5 LPM was used. Remove PIVEC from the set-up. Take out the cell culture insert and place the exposed filter in filter holder under low humidity conditions for at least 24 h prior to measurements.</li>
		<li>Clean PIVEC with 70% ethanol. Sterilize with ultraviolet light for at least 30 min prior to the next experiment.</li>
		<li>Weigh the exposed filter three times and record the filter weights. Place the exposed filter in a labeled filter holder for storage.</li>
	</ol>
	</li>
</ol>
4. Calculation of Deposited Dose and Deposition Efficiency
NOTE: Knowledge of the deposition is important for aerosol administration and interpretation of cellular response.
<ol>
	<li>Calculate deposition from mass-based measurements
	<ol>
		<li>Calculate the deposited mass on filters as the difference between the pre-exposure average weight and post-exposure average weight. This value is the mass-based deposited dose for the experiment.</li>
		<li>Use administered mass, madmin, and mass-based deposited dose determined in 4.1.1, mdep, to calculate the mass-based deposition efficiency, &#951;m, for the experiment. 
		<img alt="Equation" src="/files/ftp_upload/58916/58916eq1.jpg" /></li>
		<li>Average values from 4.1.1 and 4.1.2 for at least 3 experiments to determine deposition and deposition efficiency for PIVEC for particle size.</li>
	</ol>
	</li>
	<li>Calculate deposition from number-based measurements
	<ol>
		<li>Ensure that measurements with particle counters have been performed with counters after the PIVEC and to determine the particle concentration before the PIVEC. Integrate the particle concentration over time for the particle counter then integrate over particle diameter to determine the total particles measured.</li>
		<li>Calculate the deposited particle number as the difference between the particles administered and the particles measured post-PIVEC. This value is the number-based deposited dose for the experiment.</li>
		<li>Use administered particles, nadmin, number-based deposited dose, ndep, volumetric flow rate, V, and time, t, to calculate the number-based deposition efficiency, &#951;n, for the experiment. 
		<img alt="Equation" src="/files/ftp_upload/58916/58916eq2.jpg" /></li>
		<li>Average values from 4.2.2 and 4.2.3 for at least 3 experiments to determine deposition and deposition efficiency for PIVEC for particle size.</li>
	</ol>
	</li>
</ol>
5. Aerosol Exposure of Cells
NOTE: For the cell culture at the air-liquid interface the reader is referred to Blank et al.38 Operators should perform cell culture insert loading (steps 5.1.2-5.1.4) within a biosafety cabinet. Operators should perform aerosol exposures in a fume hood.
<ol>
	<li>Culture Cells at Air-Liquid Interface
	<ol>
		<li>Lift A549 cells from culture flask by adding trypsin-EDTA, 3 mL for a T75 flask or 1 mL for a T25 flask and incubate for 5 min at 37 &#176;C. Add 7 mL of complete media for a T75 flask or 4 mL of complete media for a T25 flask to flask and rinse flask wall with cell suspension to maximize the recovered cell number. Transfer the cell suspension to a sterile 15 mL conical tube then centrifuge cells at 800 x g for 3 min.</li>
		<li>Remove the supernatant containing trypsin-EDTA and resuspend the cell pellet in 10 mL of complete media. Remove 10 &#181;L of cell suspension and add to the hemocytometer. Count cells using a hemocytometer to determine the concentration and the total number of cells.</li>
		<li>Place 0.5 mL of complete media to each well within a 24 well plate. Place unused cell culture inserts in wells. Seed cell culture inserts on the apical side at a cell density near 1 x 105 cells/cm2 for cell types that grow at a rate near doubling per day. To seed A549 cells within a 24 well insert, seed cells at a density of 1 x 105 cells/cm2 by adding 35,000 cells to the cell culture insert. 
		NOTE: Cells with a slower growth rate can be seeded at an increased cell density.</li>
		<li>Add complete media to the apical side of cell culture insert to reach the final volume (for 24 well plate final volume is 0.25 mL).</li>
		<li>Culture for 7 days in submerged conditions, replacing media every 1-2 days. After 7 days, remove the apical media and culture for at least 1 day in ALI conditions, replacing only the basolateral media.</li>
	</ol>
	</li>
	<li>Assemble PIVEC 
	<ol>
		<li>Allow cells to equilibrate to the air-liquid interface for at least 24 h prior to exposure.</li>
		<li>Choose appropriate cell culture insert adapter for PIVEC to support the cell culture insert with the filter. Place cell culture insert adapter piece on top of PIVEC base, setting into place such that the base of the adapter piece is wider than the top. Add 4 mL of cell culture media to the well of the base of the PIVEC.</li>
		<li>Use tweezers to place the cell culture insert within adapter piece placed in step 5.2.3. Place top piece on top of adapter piece, settling into place such that the base of the top piece is wider than the top. Carefully, wrap PIVEC with a single layer of duct tape.</li>
		<li>Connect 37 mm cassette pieces on top and bottom of PIVEC, by pushing into place. Place &#188;&#8221; barbed adapters into cassette inlet and outlet.</li>
		<li>Wrap resistive heater around PIVEC such that the wires are at the base. Tape to secure.</li>
		<li>Wrap PIVEC with ~8 rounds of aluminum foil for insulation. Secure with tape.</li>
		<li>Connect short piece of 1/2&#8221; outer diameter flexible tubing to the adapter on the top of PIVEC. Remove porous tubing from sterile water and place within tubing on top of PIVEC.</li>
		<li>Place PIVEC within clamp on the ring stand and secure. Complete the set-up with the vacuum pump and aerosol set-up.</li>
	</ol>
	</li>
	<li>Expose Cells at ALI using the PIVEC
	<ol>
		<li>Use deposition efficiency determined in Step 2 to calculate the mass of particles to be aerosolized. Weigh appropriate mass and add to the aerosol system set up following step 2 within the fume hood.</li>
		<li>Expose cells by following step 2.2, in this study of biological endpoints the cells were exposed to approximately 3.5 mg of copper nanoparticles with a flow rate of 0.5 LPM and an exposure duration of 10 min. Control studies performed used humidified air to determine the influence of the air alone. Remove PIVEC from the set-up. Take out the cell culture insert, place in the sterile well plate and return to the CO2 incubator (37&#176;C, 5% CO2, 90% RH).</li>
		<li>Aspirate media from PIVEC. If performing additional experiments, rinse bottom of PIVEC with phosphate buffered solution then repeat step 5.1 and 5.2.</li>
		<li>Clean PIVEC with 70% ethanol when finished. Sterilize with ultraviolet light for at least 30 min prior to the next experiment.</li>
	</ol>
	</li>
	<li>Biological Assay Procedures 
	NOTE: Assays performed in this study were oxidative stress generation through the DCFH-DA assay and cytotoxicity through lactate dehydrogenase (LDH) release.
	<ol>
		<li>Dissolve 24.4 mg of DCFH-DA in 50 mL methanol to make 1 mM DCFH-DA solution. This solution can be stored at -20&#176;C for up to 4 months. Dilute 1 mM DCFH-DA solution by mixing 0.1 mL of 1 mM DCFH-DA solution with 9.9 mL HBSS to make 10 mL of 10 &#181;M DCFH-DA.</li>
		<li>Remove cell culture media and wash cell culture insert with approximately 1 mL of PBS. Add 0.5 mL of 10 &#181;M DCFH-DA solution to each well, replacing inserts when finished. Cover plate with aluminum foil to prevent photoactivation of the dye and return to 37&#176;C incubator for 1 h.</li>
		<li>Remove cells from the incubator and aspirate the DCFH-DA working solution from the wells. Add 0.5 mL HBSS to wells and replace cell culture inserts.</li>
		<li>Load the well plate into plate reader and measure baseline fluorescence using excitation/emission wavelengths of 485/530 nm. Remove plate from plate reader and load insert into PIVEC for exposure.</li>
		<li>Expose cells for desired exposure duration. Remove insert from PIVEC and return to well plate. Remove 50 &#181;L of basolateral fluid from well plate and place in white 96 well plate. Measure the fluorescence of DCF using excitation/emission wavelengths of 485/530 nm every 30 min post-exposure for 2 h.</li>
		<li>Let basolateral fluid equilibrate to room temperature for 20-30 min. Add 50 &#181;L of LDH assay solution, mixed following manufacturer protocol, to basolateral fluid from well plate and let react for 10 min. Add 25 &#181;L of stop solution to well. Read fluorescence of resorufin product using excitation/emission wavelengths of 560/590 nm.</li>
		<li>Remove additional basolateral fluid and repeat step 5.4.6 at 4 h and 24 h post-exposure.</li>
	</ol>
	</li>
</ol>
6 Statistical Methods
<ol>
	<li>Analysis of Biological Assay Data
	<ol>
		<li>Report ROS production as the fluorescence intensity increase of treated cells relative to baseline measurements. Report LDH activity as the fluorescence intensity increase of treated cells relative to untreated cells.</li>
		<li>Perform single factor ANOVA to determine statistical differences between data sets. Where appropriate, perform student t-tests at a value of significance of 0.05. Report data as the mean &#177; standard deviation of at least three exposure measurements.</li>
	</ol>
	</li>
</ol>

The affiliation of the authors is as shown on the cover page. The authors are financially supported by Virginia Commonwealth University, where the work was completed in Richmond, VA. The authors have sole responsibility for the writing and content of this paper. The authors declare that there are no competing interests.

Filter cassettes provide a simple, inexpensive method of collecting aerosols in the breathing zone; however, aerosol samples extracted from filters do not represent the entire aerosol (i.e. gases, volatiles, and particulates) and consequently limit the assessment of related biological effects. Using the initial design of the 37 mm filter cassette, the PIVEC is designed to maintain portability and mimic the in vivo deposition of particles from inhalation. The PIVEC is significantly smaller than current ALI exposu...

Personal sampling using in vitro techniques could provide comprehensive information regarding the biological effects of aerosols in the workplace.1 Exposures to contaminants in the air include exposures to the chemical itself, to the collected air samples, under submerged conditions where the gas is introduced to the cell suspension, intermittent exposures using a device such as a rocker, or direct exposures at the air-liquid interface (ALI).2 Many of these techniques are performed with cells grown in suspension or the collection of samples prior to the exposure, each of which can affect the toxicological study due to potential changes in the aerosol.3 To avoid these changes, the laboratory can be brought to the field using several in vitro ALI culture exposure systems that are used in literature,4,5,6,7,8,9,10,11,12,13 however, few are commercially available.8,9,12 These systems are often bulky, especially when including instruments to regulate the temperature and humidity of the cellular environment and the flow rate of the sample aerosol. By using the PIVEC, aerosol exposures can be performed outside of a traditional lab setting or within the breathing zone while mimicking inhalation conditions.
The determination of aerosol deposition in vitro is important to the investigation of health effects due to inhalation. The breathing zone, the area within 30 cm from the mouth and nose,14 is crucial for understanding the exposure to nanoparticles and for linking to the biological effects in the lungs.2 Often, the deposition on cells is defined as a deposition efficiency, the particles deposited onto and taken up by the cells divided by the particles administered to the system6,15 or on a mass basis of the same amounts.4,16 The current methods for measuring aerosols in the breathing zone are filter based, capturing particles over a given sampling period and using the filters to conduct further testing.17 Personal monitoring requires a small system that comes with the tradeoff of fewer samples.
There are many approaches to determine the health effects from exposure to an aerosol. The ALI model allows for the aerosol to be administered directly to the cells through the air as in a real exposure scenario, yet it is more cost-effective and less time intensive than in vivo studies while mimicking the air-liquid barriers such as the eyes, skin, and lungs. Lung cells grown at the ALI have the ability to generate a polarized barrier layer,18,19 which produces physiological traits that resemble the in vivo lung epithelium, including mucus and surfactant production in specific bronchial or alveolar cell lines, cilia beating,19 tight junctions,19,20 and cell polarization.18 Changes such as these can affect the cellular response measured in toxicity studies.21 In addition, ALI in vitro model results are often more sensitive than cells exposed via suspension models22 and are able to model acute in vivo inhalation toxicity.23,24 Therefore, an ALI exposure system that is able to perform measurements within the breathing zone is a natural next step.
By exposing the cells to aerosol directly at the source of emission, investigation of the effects of all gases, semi-volatile compounds, and particles involved in the mixture occurs. When the mixture is collected on a filter, the gases and volatile compounds are not captured and the whole mixture cannot be investigated. In addition, reconstitution of particles into a powder or a liquid suspension can lead to the aggregation or particle-fluid interactions, such as dissolution, in liquid suspension.25,26 When aerosol particles are added to the liquid, there is a higher potential for agglomeration,25,27 formation of a protein corona,28 or interaction with compounds in the liquid, which can affect deposition and influence the biological response.29,30
Exposure at the ALI is based on three main aerosol profiles, cloud settling, parallel flow, and perpendicular flow. Cloud settling, used by the Air-Liquid Interface Cell Exposure (ALICE),4 is a batch system where particles deposit through gravitational and diffusional settling as the aerosol is treated as one unit. Parallel flow, used by the Electrostatic Aerosol in vitro Exposure System (EAVES)5 and Multiculture Exposure Chamber (MEC) II,6 allows for deposition through the addition of Brownian motion through the flow profile. Perpendicular flow, used by a microsprayer,7 Nano Aerosol Chamber for In-Vitro Toxicity (NACIVT),11 and commercial ALI systems8,9,10,12, adds the impaction of particles within the deposition region. Many of these exposure systems are large and bulky, requiring excess systems for aerosol pre-conditioning, pumps for flow, or even heating chambers for incubation of cells. This large size decreases the portability of the system. Instead of sampling directly at the source of emission, these systems often have samples brought to the lab or model aerosols generated for analysis. The complexity of the emitted aerosol can be lost in translation from the field to the lab. The PIVEC is smaller than current systems, with an external surface area of approximately 460 cm2 and weighing only 60 grams, with thermal and humidity control incorporated into the system allowing for a highly portable device. The decreased size and weight allow the system to be worn or taken to the source of exposure, permitting direct sampling.
The large size of current exposure systems also decreases the ability to perform sampling to investigate spatial gradients in concentrations. This resolution is key when determining toxicological effects of many potential environmental and occupational hazards such as vehicular exhaust particulate matter or workplace activities where aerosolization occurs. Immediately post-emission, there becomes a spatial variance in particle concentration. This grows with time as the particles disperse throughout the atmosphere and these effects can change based on the ambient conditions, such as temperature, pressure, wind, and sun. Particles can begin to age and oxidize as well once emitted31,32 and dispersal rates are affected by the topography; higher concentrations will be found in canyons and tunnels, where dispersion effects are slowed, and lower concentrations can be found where there is a large area for dispersion.33 These changes in dispersion rates can have significant effects on human health and can be seen when comparing the number of asthmatic adults living in urban versus in rural settings.34 While many exposure systems provide multiple samples at once, multiple systems are necessary with an abundance of large equipment to perform spatial resolution.
By bringing the lab to the field, the time of analysis can be decreased by using the whole cell as a sensor. Following known biological mechanisms and endpoints can aid in the determination of the aerosol composition and size. Due to slow clearance methods, including mucociliary clearance, phagocytosis, and translocation, these particles are often interacting with cells for approximately days to weeks3 generating oxidative stress, inflammation, and even cell death. These biological endpoints can be the starting points for adverse outcome pathways for cardiovascular disease or chronic obstructive pulmonary disease. In addition, Wiemenn et al. performed an array of in vitro assays to compare with literature values for short term in vivo inhalation toxicity.35&#160;In vivo response was predicted with two of four positive results from testing cytotoxicity via lactate dehydrogenase release, oxidative stress from glutathione reduction and hydrogen peroxide formation and release, and inflammation potential from the tumor necrosis factor alpha gene. Out of ten nanosized metal oxides tested, six tested as active (titanium oxide, zinc oxide, and four different cerium oxide) using exposures in vitro with confirmation in vivo.
In order to study the effects of aerosols in an occupational setting, our lab developed the PIVEC for exposures in the field. Additionally, the PIVEC can be worn for personal sampling to monitor and investigate inhalation exposure like the 37 mm filter cassette36 or multiple systems can be used to achieve spatial resolution within a given area. In this protocol, the characterization and use of the PIVEC is discussed. After exposure, the biological effects are observed through cytotoxicity assays.

<table><tbody><tr><td>Scanning mobility particle sizer (SMPS)</td><td>TSI, Inc.</td><td>3910</td><td>NanoSMPS</td></tr><tr><td>Optical particle sizer (OPS)</td><td>TSI, Inc.</td><td>3330</td><td></td></tr><tr><td>Stainless Steel Pipe, 4&quot; Long</td><td>McMaster-Carr</td><td>4830K116</td><td>Standard-Wall 304/304L, Threaded on Both Ends, 1/8 Pipe Size</td></tr><tr><td>Brass Ball Valve with Lever Handle</td><td>McMaster-Carr</td><td>4112T12</td><td>Compact High-Pressure Rating, 1/8 NPT Female</td></tr><tr><td>Steel Pipe, 2&quot; Long</td><td>McMaster-Carr</td><td>7753K121</td><td>Standard Wall, Threaded on One End, 1/8 Pipe Size</td></tr><tr><td>HEPA filter</td><td>GE Healthcare</td><td>09-744-12</td><td>HEPA-Cap Disposable Air Filtration Capsule</td></tr><tr><td>Vacuum Generator</td><td>PISCO USA</td><td>VCH10-018C</td><td></td></tr><tr><td>PIVEC</td><td>VCU</td><td></td><td>For design please contact authors</td></tr><tr><td>Resistive heater</td><td></td><td></td><td></td></tr><tr><td>1/4&quot; barbed connectors</td><td>Zefon International, Inc.</td><td>459743</td><td></td></tr><tr><td>Porous tubing</td><td>Scientific Commodities, Inc.</td><td>BB2062-1814A</td><td>Hydrophilic 10 um pores</td></tr><tr><td>Battery power bank</td><td></td><td></td><td></td></tr><tr><td>Cell culture insert</td><td>Fisherbrand</td><td>353095</td><td>24 well plate insert</td></tr><tr><td>Filter Forceps</td><td>Fisherbrand</td><td>09-753-50</td><td></td></tr><tr><td>Transfer Pipette</td><td>ThermoScientific</td><td>13-711-27</td><td></td></tr><tr><td>Glass Fiber Filters</td><td>SKC</td><td>225-7</td><td>Binder-Free Type AE Filter 37 MM 1.00 um pore</td></tr><tr><td>Ultra Micro Balance</td><td>A&amp;amp;D</td><td>BM-22</td><td>Housed in environmental chamber</td></tr><tr><td>37 mm filter cassette</td><td>SKC</td><td>225-3250</td><td>Filter Cassette Blank, 37 mm, Clear Styrene</td></tr><tr><td>Variable flow vacuum pump</td><td>SKC</td><td>220-5000TC</td><td>AirChek TOUCH, 5 to 5000 mL/min</td></tr><tr><td>Copper Particles</td><td>U.S. Research Materials, Inc.</td><td>US1090</td><td>40 nm</td></tr><tr><td>Copper Particles</td><td>U.S. Research Materials, Inc.</td><td>US1088</td><td>100 nm</td></tr><tr><td>Copper Particles</td><td>U.S. Research Materials, Inc.</td><td>US1117M</td><td>800 nm</td></tr></tbody></table>

a new portable in vitro exposure cassette for aerosol sampling

This protocol introduces a new in vitro exposure system, capable of being worn, including its characterization and performance. Air-liquid interface (ALI) in vitro exposure systems are often large and bulky, making transport to the field and operation at the source of emission or within the breathing zone difficult. Through miniaturization of these systems, the lab can be brought to the field, expediting processing time and providing a more appropriate exposure method that does not alter the aerosol prior to contacting the cells. The Portable In vitro Exposure Cassette (PIVEC) adapts a 37 mm filter cassette to allow for in vitro toxicity testing outside of a traditional laboratory setting. The PIVEC was characterized using three sizes of copper nanoparticles to determine deposition efficiency based on gravimetric and particle number concentration analysis. Initial cytotoxicity experiments were performed with exposed lung cells to determine the ability of the system to deposit particles while maintaining cell viability. The PIVEC provides a similar or increased deposition efficiency when comparing to available perpendicular flow in vitro exposure devices. Despite the lower sample throughput, the small size gives some advantages to the current in vitro ALI exposure systems. These include the ability to be worn for personal monitoring, mobility from the laboratory to the source of emission, and the option to set-up multiple systems for spatial resolution while maintaining a lower user cost. The PIVEC is a system capable of collecting aerosols in the field and within the breathing zone onto an air-interfaced, in vitro model.

Occupational in vitro toxicology involves maintaining cellular viability while performing aerosol exposure. The PIVEC system is shown in Figure 2, including the temperature and humidity control and the worn PIVEC. The temperature was maintained using a battery-powered resistive heater and the aerosol humidified using increased natural humidification through a porous, wetted tube. In a controlled aerosol setting inside a laboratory, the PIVEC can be s...

Watch this Scientific Journal Video about A New Portable In Vitro Exposure Cassette for Aerosol Sampling at JoVE.com

A New Portable In Vitro Exposure Cassette for Aerosol Sampling

Here, we present a protocol to perform portable cellular aerosol exposures and measure cellular response. The method uses cells, grown at the air-liquid interface, mimicking in vivo physiology. Cellular response to copper nanoparticle aerosols was observed as oxidative stress through reactive oxygen species generation and cytotoxicity as lactate dehydrogenase release.

A New Portable In Vitro Exposure Cassette for Aerosol Sampling

This protocol introduces a new in vitro exposure system, capable of being worn, including its characterization and performance. Air-liquid interface (ALI) ...

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7.1K Views.  Virginia Commonwealth University. Here, we present a protocol to perform portable cellular aerosol exposures and measure cellular response. The method uses cells, grown at the air-liquid interface, mimicking in vivo physiology. Cellular response to copper nanoparticle aerosols was observed as oxidative stress through reactive oxygen species generation and cytotoxicity as lactate dehydrogenase release.

Video: A New Portable In Vitro Exposure Cassette for Aerosol Sampling

Collection and Extraction of Occupational Air Samples for Analysis of Fungal DNA

Traditional methods of identifying fungal exposures in occupational environments, such as culture and microscopy-based approaches, have several limitations that have resulted in the exclusion of many species. Advances in the field over the last two decades have led occupational health researchers to turn to molecular-based approaches for identifying fungal hazards. These methods have resulted in the detection of many species within indoor and occupational environments that have not been detected using traditional methods. This protocol details an approach for determining fungal diversity within air samples through genomic DNA extraction, amplification, sequencing, and taxonomic identification of fungal internal transcribed spacer (ITS) regions. ITS sequencing results in the detection of many fungal species that are either not detected or difficult to identify to species level using culture or microscopy. While these methods do not provide quantitative measures of fungal burden, they offer a new approach to hazard identification and can be used to determine overall species richness and diversity within an occupational environment.

Determining the fungal diversity within an environment is a method utilized in occupational health studies to identify health hazards. This protocol describes DNA extraction from occupational air samples for amplification and sequencing of fungal ITS regions. This approach detects many fungal species that can be overlooked by traditional assessment methods.

Traditional methods of identifying fungal exposures in occupational environments, such as culture and microscopy-based approaches, have several ...

Impact Assessment of Repeated Exposure of Organotypic 3D Bronchial and Nasal Tissue Culture Models to Whole Cigarette Smoke

Cigarette smoke (CS) has a major impact on lung biology and may result in the development of lung diseases such as chronic obstructive pulmonary disease or lung cancer. To understand the underlying mechanisms of disease development, it would be important to examine the impact of CS exposure directly on lung tissues. However, this approach is difficult to implement in epidemiological studies because lung tissue sampling is complex and invasive. Alternatively, tissue culture models can facilitate the assessment of exposure impacts on the lung tissue. Submerged 2D cell cultures, such as normal human bronchial epithelial (NHBE) cell cultures, have traditionally been used for this purpose. However, they cannot be exposed directly to smoke in a similar manner to the in vivo exposure situation. Recently developed 3D tissue culture models better reflect the in vivo situation because they can be cultured at the air-liquid interface (ALI). Their basal sides are immersed in the culture medium; whereas, their apical sides are exposed to air. Moreover, organotypic tissue cultures that contain different type of cells, better represent the physiology of the tissue in vivo. In this work, the utilization of an in vitro exposure system to expose human organotypic bronchial and nasal tissue models to mainstream CS is demonstrated. Ciliary beating frequency and the activity of cytochrome P450s (CYP) 1A1/1B1 were measured to assess functional impacts of CS on the tissues. Furthermore, to examine CS-induced alterations at the molecular level, gene expression profiles were generated from the tissues following exposure. A slight increase in CYP1A1/1B1 activity was observed in CS-exposed tissues compared with air-exposed tissues. A network-and transcriptomics-based systems biology approach was sufficiently robust to demonstrate CS-induced alterations of xenobiotic metabolism that were similar to those observed in the bronchial and nasal epithelial cells obtained from smokers.

The aim of this protocol is to expose human organotypic 3D bronchial and nasal tissue models to mainstream cigarette smoke (CS) at the air-liquid interface. The impact of CS on the tissues is then investigated using a cytochrome P450 activity assay, a cilia beating measurement, and a systems biology approach.

Cigarette smoke (CS) has a major impact on lung biology and may result in the development of lung diseases such as chronic obstructive pulmonary disease ...

Bioengineering

Generation of Electronic Cigarette Aerosol by a Third-Generation Machine-Vaping Device: Application to Toxicological Studies

Electronic-cigarette (e-cig) devices use heat to produce an inhalable aerosol from a liquid (e-liquid) composed mainly of humectants, nicotine, and flavoring chemicals. The aerosol produced includes fine and ultrafine particles, and potentially nicotine and aldehydes, which can be harmful to human health. E-cig users inhale these aerosols and, with the third-generation of e-cig devices, control design features (resistance and voltage) in addition to the choice of e-liquids, and the puffing profile. These are key factors that can significantly impact the toxicity of the inhaled aerosols. E-cig research, however, is challenging and complex mostly due to the absence of standardized assessments and to the numerous varieties of e-cig models and brands, as well as e-liquid flavors and solvents that are available on the market. These considerations highlight the urgent need to harmonize e-cig research protocols, starting with e-cig aerosol generation and characterization techniques. The current study focuses on this challenge by describing a detailed step-by-step e-cig aerosol generation technique with specific experimental parameters that are thought to be realistic and representative of real-life exposure scenarios. The methodology is divided into four sections: preparation, exposure, post-exposure analysis, plus cleaning and maintenance of the device. Representative results from using two types of e-liquid and various voltages are presented in terms of mass concentration, particle size distribution, chemical composition and cotinine levels in mice. These data demonstrate the versatility of the e-cig exposure system used, aside from its value for toxicological studies, as it allows for a broad range of computer-controlled exposure scenarios, including automated representative vaping topography profiles.

Electronic cigarette (e-cig) users are increasing worldwide. Little, however, is known about the health effects induced by inhaled e-cig aerosols. This article describes an e-cig aerosol generation technique suitable for animal exposures and subsequent toxicological studies. Such protocols are required to establish experimentally reproducible and standardized e-cig exposure systems.

Electronic-cigarette (e-cig) devices use heat to produce an inhalable aerosol from a liquid (e-liquid) composed mainly of humectants, nicotine, and ...

Chemistry

Development of a Nose-only Inhalation Toxicity Test Chamber That Provides Four Exposure Concentrations of Nano-sized Particles

Using a numerical analysis based on computerized fluid dynamics, a nose-only inhalation toxicity chamber with four different exposure concentrations is designed and validated for flow field uniformity and cross-contamination among the exposure ports for each concentration. The designed flow field values are compared with the measured values from exposure ports located horizontally and vertically. For this purpose, nanoscale sodium chloride particles are generated as test particles and introduced to the inhalation chamber to evaluate the cross-contamination and concentration maintenance among the chambers, for each concentration group. The results indicate that the designed multiconcentration inhalation chamber can be used in animal inhalation toxicity testing without cross-contamination among concentration groups. Moreover, the designed multiconcentration inhalation toxicity chamber can also be converted to a single-concentration inhalation chamber. Further testing with gas, organic vapor, or non-nanoscale particles will ensure the use of the chamber in the inhalation testing of other test articles.

A nose-only inhalation toxicity chamber capable of testing inhalation toxicity at four different exposure concentrations was designed and validated for flow field uniformity and cross-contamination between the exposure ports for each concentration. Here, we present a protocol to confirm that the designed chamber is effective for inhalation toxicity testing.

Using a numerical analysis based on computerized fluid dynamics, a nose-only inhalation toxicity chamber with four different exposure concentrations is ...

Engineering

Use of Capillary Aerosol Generator in Continuous Production of Controlled Aerosol for Non-Clinical Studies

The capillary aerosol generator (CAG) is operated with the principal of thermal liquid evaporation through heating of e-liquid in the initial phase, followed by nucleation and condensation regulated through a mixture of airflow to generate aerosols, such as in an electronic cigarette (EC). The CAG is particularly useful in generating aerosols of large volumes in a continuous manner, for instances such as in vivo inhalation toxicology studies, where usage of ECs is not feasible. The thermal effects of generating aerosol from the CAG are similar in terms of temperature applied in an EC, thus allowing investigators to assess the vapors of e-liquids at scale and reproducibility. As the operation of the CAG allows users to control critical parameters such as the flow rate of e-liquid, heating temperatures and dilution air flows, it allows investigators to test various e-liquid formulations in a well-controlled device. Properties, such as aerosol particle size, are demonstrated to be regulated with the air flow rate with respect to the e-liquid flow and e-liquid composition. The CAG, however, is limited in assessing common EC-related issues, such as overheating of its elements. We seek to demonstrate that the CAG can generate aerosol that is reproducible and continuous, by assessing the chemical and physical aerosol characteristics with a chosen e-liquid formulation. The protocol describes the operating parameters of liquid flow rate, dilution air-flow rates and operating procedures needing to optimize the aerosol concentration and particle size required for an in vivo toxicology study. Presenting the representative results from the protocol and discussing the challenges and applications of working with a CAG, we demonstrate that CAG can be used in a reproducible fashion. The technology and protocol, that has been developed from prior work, serve as a foundation for future innovations for laboratory-controlled aerosol generation investigations.

The protocol describes the settings and use of a capillary aerosol generator for continuous production of controlled aerosol from a multispecies liquid solution, suitable for steady large-volume aerosol delivery (e.g., in vivo inhalation studies).

The capillary aerosol generator (CAG) is operated with the principal of thermal liquid evaporation through heating of e-liquid in the initial phase, ...

Dry Powder and Nebulized Aerosol Inhalation of Pharmaceuticals Delivered to Mice Using a Nose-only Exposure System

Obstructive respiratory diseases like asthma and chronic obstructive pulmonary disease (COPD) are currently treated by inhaled anti-inflammatory and bronchodilator drugs. Despite the availability of multiple treatments, both diseases are growing public health concerns. The majority of asthma patients are well controlled on current inhaled therapies but a substantial number of patients with severe asthma are not. Asthma affects an estimated 300 million people worldwide and approximately 20 percent have a severe form of the disease. In contrast to asthma, there are few effective therapies for COPD. An estimated 10% of the population has COPD and the trend in death rates is increasing for COPD while decreasing for other major diseases. Although developing drugs for inhaled delivery is challenging, the nose-only inhalation unit enables direct delivery of novel drugs to the lung of rodents for pre-clinical efficacy and safety/toxicology studies. Inhaled drug delivery has multiple advantages for respiratory diseases, where high concentration in the lung improves efficacy and minimizes systemic side effects. Inhaled corticosteroids and bronchodilators benefit from these advantages and inhaled delivery may also hold potential for future biologic therapies. The inhalation unit described herein can generate, sample for characterization, and uniformly deposit a drug aerosol in the lungs of rodents. This enables the pre-clinical determination of the efficacy and safety of drug doses deposited in the lungs of rodents, key data required before initiating clinical development.

The inhalation unit described herein can generate, sample for characterization, and uniformly deposit a drug aerosol in the lungs of rodents. This enables the pre-clinical determination of the efficacy and safety of drug doses deposited in the lungs; key data enabling clinical inhaled drug development.

Obstructive respiratory diseases like asthma and chronic obstructive pulmonary disease (COPD) are currently treated by inhaled anti-inflammatory and ...

Medicine

Whole-Body Nanoparticle Aerosol Inhalation Exposures

Inhalation is the most likely exposure route for individuals working with aerosolizable engineered nano-materials (ENM). To properly perform nanoparticle inhalation toxicology studies, the aerosols in a chamber housing the experimental animals must have: 1) a steady concentration maintained at a desired level for the entire exposure period; 2) a homogenous composition free of contaminants; and 3) a stable size distribution with a geometric mean diameter &lt; 200 nm and a geometric standard deviation &sigma;g &lt; 2.5 5. The generation of aerosols containing nanoparticles is quite challenging because nanoparticles easily agglomerate. This is largely due to very strong inter-particle forces and the formation of large fractal structures in tens or hundreds of microns in size 6, which are difficult to be broken up. Several common aerosol generators, including nebulizers, fluidized beds, Venturi aspirators and the Wright dust feed, were tested; however, none were able to produce nanoparticle aerosols which satisfy all criteria 5.
A whole-body nanoparticle aerosol inhalation exposure system was fabricated, validated and utilized for nano-TiO2 inhalation toxicology studies. Critical components: 1) novel nano-TiO2 aerosol generator; 2) 0.5 m3 whole-body inhalation exposure chamber; and 3) monitor and control system. Nano-TiO2 aerosols generated from bulk dry nano-TiO2 powders (primary diameter of 21 nm, bulk density of 3.8 g/cm3) were delivered into the exposure chamber at a flow rate of 90 LPM (10.8 air changes/hr). Particle size distribution and mass concentration profiles were measured continuously with a scanning mobility particle sizer (SMPS), and an electric low pressure impactor (ELPI). The aerosol mass concentration (C) was verified gravimetrically (mg/m3). The mass (M) of the collected particles was determined as M = (Mpost-Mpre), where Mpre and Mpost are masses of the filter before and after sampling (mg). The mass concentration was calculated as C = M/(Q*t), where Q is sampling flowrate (m3/min), and t is the sampling time (minute). The chamber pressure, temperature, relative humidity (RH), O2 and CO2 concentrations were monitored and controlled continuously. Nano-TiO2 aerosols collected on Nuclepore filters were analyzed with a scanning electron microscope (SEM) and energy dispersive X-ray (EDX) analysis.
In summary, we report that the nano-particle aerosols generated and delivered to our exposure chamber have: 1) steady mass concentration; 2) homogenous composition free of contaminants; 3) stable particle size distributions with a count-median aerodynamic diameter of 157 nm during aerosol generation. This system reliably and repeatedly creates test atmospheres that simulate occupational, environmental or domestic ENM aerosol exposures.

A whole-body nanoparticle aerosol inhalation exposure facility was constructed for nano-sized titanium dioxide (TiO2) inhalation toxicology studies. This system provides nano-TiO2 aerosol test atmospheres that have: 1) a steady mass concentration; 2) a homogenous composition free of contaminants; and 3) a stable particle size distribution during aerosol generation.

Inhalation is the most likely exposure route for individuals working with aerosolizable engineered nano-materials (ENM). To properly perform nanoparticle ...

Biology

Cigarette Smoke Exposure in Mice using a Whole-Body Inhalation System

Close to 14% of adults in the United States were reported to smoke cigarettes in 2018. The effects of cigarette smoke (CS) on lungs and cardiovascular diseases have been widely studied, however, the impact of CS in other tissues and organs such as blood and bone marrow remain incompletely defined. Finding the appropriate system to study the effects of CS in rodents can be prohibitively expensive and require the purchase of commercially available systems. Thus, we set out to build an affordable, reliable, and versatile system to study the pathologic effects of CS in mice. This whole-body inhalation exposure system (WBIS) set-up mimics the breathing and puffing of cigarettes by alternating exposure to CS and clean air. Here we show that this do-it-yourself (DIY) system induces airway inflammation and lung emphysema in mice after 4-months of cigarette smoke exposure. The effects of whole-body inhalation (WBI) of CS on hematopoietic stem and progenitor cells (HSPCs) in the bone marrow using this apparatus are also shown.

This protocol demonstrates the study of the pathophysiologic effects of cigarette smoke (CS) with a whole-body inhalation (WBI) exposure system (WBIS) built in-house. This system can expose animals to CS under controlled repeatable conditions for research of CS-mediated effects on lung emphysema and hematopoiesis.

Close to 14% of adults in the United States were reported to smoke cigarettes in 2018. The effects of cigarette smoke (CS) on lungs and cardiovascular ...

Immunology and Infection

Assessment of the Acute Inhalation Toxicity of Airborne Particles by Exposing Cultivated Human Lung Cells at the Air-Liquid Interface

Here, we present a specially designed modular in vitro exposure system that enables the homogenous exposure of cultivated human lung cells at the ALI to gases, particles or complex atmospheres (e.g., cigarette smoke), thus providing realistic physiological exposure of the apical surface of the human alveolar region to air. In contrast to sequential exposure models with linear aerosol guidance, the modular design of the radial flow system meets all requirements for the continuous generation and transport of the test atmosphere to the cells, a homogenous distribution and deposition of the particles and the continuous removal of the atmosphere. This exposure method is primarily designed for the exposure of cells to airborne particles, but can be adapted to the exposure of liquid aerosols and highly toxic and aggressive gases depending on the aerosol generation method and the material of the exposure modules.
Within the framework of a recently completed validation study, this exposure system was proven as a transferable, reproducible and predictive screening method for the qualitative assessment of the acute pulmonary cytotoxicity of airborne particles, thereby potentially reducing or replacing animal experiments that would normally provide this toxicological assessment.

We present a robust, transferable and predictive in vitro exposure system for the screening and monitoring of airborne particles concerning their acute pulmonary cytotoxicity by exposing cultivated human lung cells at the air-liquid interface (ALI).

Here, we present a specially designed modular in vitro exposure system that enables the homogenous exposure of cultivated human lung cells at the ALI to ...

An Air-liquid Interface Bronchial Epithelial Model for Realistic, Repeated Inhalation Exposure to Airborne Particles for Toxicity Testing

For toxicity testing of airborne particles, air-liquid interface (ALI) exposure systems have been developed for in vitro tests in order to mimic realistic exposure conditions. This puts specific demands on the cell culture models. Many cell types are negatively affected by exposure to air (e.g., drying out) and only remain viable for a few days. This limits the exposure conditions that can be used in these models: usually relatively high concentrations are applied as a cloud (i.e., droplets containing particles, which settle down rapidly) within a short period of time. Such experimental conditions do not reflect realistic long-term exposure to low concentrations of particles. To overcome these limitations the use of a human bronchial epithelial cell line, Calu-3 was investigated. These cells can be cultured at ALI conditions for several weeks while retaining a healthy morphology and a stable monolayer with tight junctions. In addition, this bronchial model is suitable for testing the effects of repeated exposures to low, realistic concentrations of airborne particles using an ALI exposure system. This system uses a continuous airflow in contrast to other ALI exposure systems that use a single nebulization producing a cloud. Therefore, the continuous flow system is suitable for repeated and prolonged exposure to airborne particles while continuously monitoring the particle characteristics, exposure concentration, and delivered dose. Taken together, this bronchial model, in combination with the continuous flow exposure system, is able to mimic realistic, repeated inhalation exposure conditions that can be used for toxicity testing.

Described is the cell culture and exposure method of an in vitro bronchial model for realistic, repeated inhalation exposure to particles for toxicity testing.

For toxicity testing of airborne particles, air-liquid interface (ALI) exposure systems have been developed for in vitro tests in order to mimic realistic ...

A New Portable In Vitro Exposure Cassette for Aerosol Sampling

Summary

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Collection and Extraction of Occupational Air Samples for Analysis of Fungal DNA

Impact Assessment of Repeated Exposure of Organotypic 3D Bronchial and Nasal Tissue Culture Models to Whole Cigarette Smoke

Generation of Electronic Cigarette Aerosol by a Third-Generation Machine-Vaping Device: Application to Toxicological Studies

Development of a Nose-only Inhalation Toxicity Test Chamber That Provides Four Exposure Concentrations of Nano-sized Particles

Use of Capillary Aerosol Generator in Continuous Production of Controlled Aerosol for Non-Clinical Studies

Dry Powder and Nebulized Aerosol Inhalation of Pharmaceuticals Delivered to Mice Using a Nose-only Exposure System

Whole-Body Nanoparticle Aerosol Inhalation Exposures

Cigarette Smoke Exposure in Mice using a Whole-Body Inhalation System

Assessment of the Acute Inhalation Toxicity of Airborne Particles by Exposing Cultivated Human Lung Cells at the Air-Liquid Interface

An Air-liquid Interface Bronchial Epithelial Model for Realistic, Repeated Inhalation Exposure to Airborne Particles for Toxicity Testing