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, ...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#34; 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&#34; 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&#34; 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&#38;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) ...

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.

a-new-portable-in-vitro-exposure-cassette-for-aerosol-sampling

Research

JoVE Journal

Environment

7.0K visualizações.  Virginia Commonwealth University. Este protocolo é significativo, pois o PIVEC permite a exposição direta de uma cultura celular a aerossóis do mundo real. O sistema pode ser avisado para provar o mesmo ar que uma pessoa respira, permitindo um novo tipo de monitoramento da qualidade do ar. A principal vantagem dessa técnica é a facilidade de capturar aerossóis em células pulmonares cultivadas em locais inacessíveis para sistemas de bancada, como na fonte de admissão ou dentro da zona respiratória.Demonstrand...