This work was supported by the German Federal Ministry of Education and Research (Bundesministerium f&#252;r Bildung und Forschung, BMBF, Germany (Grant 031A581, sub-project A-D)) and by the German Research Foundation (Deutsche Forschungsgesellschaft, DFG, Research Training Group GRK 2338).

NOTE: The protocol of one exposure experiment covers a period of three days.
Day 1
1. General preparations and cultivation of cells
NOTE: The human lung adenocarcinoma epithelial cell line A549 was used for exposure experiments. Cells must be handled under sterile conditions. Other cell lines that are suitable for cultivation at the ALI can be used.
<ol>
	<li>Prepare the growth medium (Dulbecco&#39;s Minimum Essential Medium (DMEM), supplemented with 10% fetal bovine serum (FBS) and 5 &#181;g/mL gentamycin) and the exposure medium (DMEM, supplemented with 5 &#181;g/mL gentamycin and a final HEPES concentration of 100 mM).</li>
	<li>Culture A549 cells in growth medium at 37 &#176;C in a humidified atmosphere containing 5% CO2.</li>
	<li>Cultivate cells in cell culture flask of 75 cm2 (T-75) in 14 mL of growth medium until a confluence of 80-90% before splitting (every 2-3 days) and a passage of 35.</li>
	<li>Calculate the suspension volume and the required number of cell culture inserts (three cell culture inserts as incubator controls and cell culture inserts for clean air and particle exposure) and cell culture plates.</li>
	<li>Before trypsinization and seeding of cells, add 2.5 mL of tempered growth medium to each well of a 6-well plate. Place the cell culture inserts without cells carefully inside the wells and add 1 mL growth medium to every cell culture insert. Incubate the 6-well plates for at least 30 min in the incubator (37 &#176;C, 5% CO2).</li>
</ol>
2. Trypsinization of cells
<ol>
	<li>Temper phosphate buffered saline (PBS) and growth medium at 37 &#176;C and the trypsin/EDTA (0.05%/ 0.02%) solution at room temperature.</li>
	<li>Aspirate the cell culture medium from the cell culture flask and wash the cells carefully with 8 mL of pre-heated PBS.</li>
	<li>Remove the PBS, add 2 mL of the trypsin/EDTA (0.05%/ 0.02%) solution to the cells and incubate for maximal 6 min. in the incubator at 37 &#176;C. Control the detachment process under the microscope.</li>
	<li>Neutralize the trypsin activity by adding 8 mL pre-heated growth medium, detach the cells by gently tapping sideways on the flask and resuspend the cells by repeated pipetting up and down.</li>
	<li>Transfer the cell suspension to a 50 mL tube. Determine the cell number for further procedure (e.g., seeding of cells, passage of cells).</li>
</ol>
3. Determination of cell number
NOTE: Cell concentration was determined using a cell counter or counting chambers.
<ol>
	<li>Dilute 100 &#181;L of the cell culture suspension in a cup filled with 10 mL of isotonic solution. Tilt the cup slowly without shaking.</li>
	<li>Determine number of viable cells/mL and cell viability based on the cell-specific measurement parameters of A549 cells.</li>
</ol>
4. Seeding of cells onto microporous membranes in cell culture inserts
NOTE: The exposure system is equipped with special adapters to enable the use of commercial inserts from different suppliers and of different sizes. For these exposure experiments, 6-well plates and the corresponding cell culture inserts were used. All working steps have to be done under sterile conditions.
<ol>
	<li>Provide pre-heated growth medium under sterile cell culture conditions (laminar flow).</li>
	<li>Prepare a sufficiently high volume of the cell suspension with a cell concentration of 3.0 x 105 cells/mL.</li>
	<li>After tempering the plates for 30 min, aspirate the medium within the cell culture inserts and seed 1 mL of A549 cells with a density of 3.0 x 105 cells/mL in each cell culture insert. Distribute the cell suspension by gentle rocking.</li>
	<li>Incubate the cell culture inserts with the cell suspension for 24 h (37 &#176;C and 5% CO2).</li>
</ol>
5. Pressing of test substances
NOTE: Test substances were pressed into powder cakes using a fully controllable hydraulic press. The press package can apply a maximum force of 18 kN, which is displayed as the current oil pressure (in bar) of the press package. Press conditions (pressing pressure, time of pressing) of unknown test substances have to be established and characterized in preliminary tests. Depending on the press properties of a substance, different pressing parameters and kinds of pressing plunger can be used.
CAUTION: Wear protective equipment when pressing toxic or dangerous substances.
<ol>
	<li>Set the pressing time via the time control on the front side of the press.</li>
	<li>Open the compressed air supply at the compressed air valve. Set the compressed air pressure to approximately 2 bar (indicated by a pressure gauge on the front side) using the pressure regulator on the front side of the press or on the compressed air valve of the compressed air supply. Pull out the drawer, press the Press button and read the pressing pressure on the digital pressure switch.
	<ol>
		<li>Read just the pressure at the pressure regulator if the pressure is too high or too low.</li>
	</ol>
	</li>
	<li>Assembly the substance container and ensure that the glass cylinder is correctly centered (Supplementary Figure 1). 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.</li>
	<li>Place the substance container with the plunger in the drawer and press the Press button. The hydraulic piston of the press moves onto the plunger and exerts a pressure on the test substance for the set pressing time. Open the drawer and remove the plunger.</li>
	<li>Repeat steps 5.3 and 5.4 until the substance container is at least half full.</li>
	<li>After completion of the pressing work, remove the substance container from the drawer and turn it upside down to remove loose and deposited particles.</li>
	<li>If the substance container is not needed at the same day, close the substance container with parafilm in order to prevent the test substance from drying out or absorbing moisture.</li>
</ol>
Day 2
6. Assembly of the exposure system and connecting the peripheral equipment
NOTE: A more detailed view is provided in Figure 3, Supplementary Figure 2 and Supplementary Figure 3. Assemble both modules and the aerosol generator according to the manufacturer&#39;s instructions.
<ol>
	<li>Place the exposure system on a solid and even surface, with the water supply facing forward. Connect the mass flow controllers with the aerosol generator and a three-necked bottle connected to the module for clean air exposure.</li>
	<li>Connect the flow controller and the vacuum pump. Connect the tubes from the flow controllers with the tube connector on the attachments of the aerosol guiding module. Connect the tubes on the other side of the flow controllers with the vacuum pump. Make sure that the flow is going from the module through the flow controllers to the vacuum pump.</li>
	<li>Connect the water bath with the heating water supply. The water supply is going from the water bath to the water inlet on the aerosol guiding module. Connect the water outlet of the aerosol guiding module with the water inlet of the sampling module. Close the circle with a connection from the water outlet of the sampling module to the water bath.</li>
	<li>Place the aerosol generator including the elutriator close to the exposure module and connect the excess lines of the elutriator and the exposure and clean air module with large micro filters, and the suction of the exposure chambers with small micro filters (e.g., disposable filters). The elutriator serves as a reservoir for the generated particulate atmosphere and retains particles bigger than approx. 7 &#181;m, whereas smaller particles are transported to the exposure module.</li>
	<li>Connect the computer used to control the aerosol generation to the USB port of the aerosol generator top via a USB cable and the power supply to the power supply port. Connect the AC power plug of the power supply unit to a socket (220-240 V).</li>
	<li>Connect the pipes for the medium supply and removal with two pumps. Instead of using a pump for the medium supply, the medium can also be filled manually.</li>
</ol>
7. Preparation for clean air and particle exposure
<ol>
	<li>Turn on the vacuum pump, the flow controllers and the water bath (37 &#176;C) for a warm-up period of at least 30 min.</li>
	<li>Open the compressed air supply. Set the mass flow controllers to 8 L/min for the supply line to the aerosol generator and to 3 L/min for the supply line to the three-necked bottle. Close the tabs of the mass flow controllers. 
	NOTE: These values may vary depending on the characteristics of the test substance.</li>
	<li>Adjust the flow controllers via the computer to regulate the module flow (1.5 L/min) and the chamber suction (30 mL/min).</li>
</ol>
8. Leakage test of the radial flow system
NOTE: The leakage check must be performed under vacuum and for both modules (exposure and clean air module) in order to ensure that the module has been reassembled properly.
<ol>
	<li>Remove the inlet adapter 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.</li>
	<li>Connect the vacuum lines without the filter with the tube connector of the aerosol guiding module. Close the module by using the hand wheel and measure the value of the flow controllers. The values should decrease some minutes after closing below 5 mL/min.</li>
	<li>After the impermeability check, remove all plugs and dummy flaps, insert the inlet adapter and the condensate reflector into the aerosol guiding module and connect the pipes for medium supply and removal.</li>
</ol>
9. Aerosol generation
<ol>
	<li>Start the computer and the software (Supplementary Figure 4). Start the aerosol generator software by double clicking on the aerosol generator start button on the desktop of the computer. A message window appears and asks if the settings should be reset or not. Click Yes if the software is started for the first time that day. Set the values for Slide Position and Scraper Position to the default values. Click No to keep the values for Slide Position and Scraper Position or the slide is not in the starting position.</li>
	<li>Screw a substance scraper into the pipe, which is located in the central opening of the aerosol generator top. 
	NOTE: Depending on the press characteristics, distinct types of substance scraper can be used.</li>
	<li>Use the button Homing Mode if the substance scraper is not in the lowest position.</li>
	<li>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. Make sure that the two holes in the substance container fit onto the two pins of the aerosol generator top. Place the locking plate in the slot over the substance container and tighten the black screw.</li>
	<li>Change the values for Feed (0.24 to 20 mm/h) and Rotation (1 to 800 revs/h) to the desired settings. The particle concentration can be modified by increasing or decreasing the Feed value or the carrier gas flow rate.</li>
	<li>Use the downward arrows to push down the slide with the substance container until the substance scraper is near the pressed substance.</li>
	<li>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 15-20 mm/h to avoid long waiting times.</li>
	<li>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). Change the value for Feed back to the desired settings when the first aerosol vapor reaches continuously the Elutriator and click on the Stop button.</li>
</ol>
10. Exposure experiments
<ol>
	<li>Start the medium supply with pre-heated exposure medium and fill the sampling modules until the downpipes are covered while the module is open. Use the medium pump or fill the medium manually (25 mL per individual exposure chamber).</li>
	<li>Insert blind cell culture inserts (inserts without cells) 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.</li>
	<li>Start the aerosol generator, close the exposure module and connect the exposure module to the exposure module outlet of the aerosol generator. Give the aerosol generator a lead time of at least 20-30 min before exposures are started in order to enable a stable generation of particles.</li>
	<li>Prepare the post-incubation plates for the incubator controls and the exposed cell culture inserts during the lead time. Add 1.5 mL of growth medium per well and incubate the plates in the incubator (37 &#176;C, 5% CO2).</li>
	<li>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 (using the pump or manually) until the downpipes are covered with medium. Now, open also the compressed air supply to the clean air module with the tap of the mass flow controller (3 L/min)</li>
	<li>Remove the cell culture inserts from the 6-well plates with the help of a tweezer. Pour the growth medium carefully from the cell culture inserts off by toppling the inserts and aspirate and discard the residual liquid using a pipette. Place the inserts in the exposure chambers of both modules, the exposure and clean air module.</li>
	<li>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. 
	NOTE:The particle concentration can be modified by increasing/decreasing the Feed value, the carrier gas flow rate or the time of exposure.</li>
	<li>Disconnect the exposure and clean air modules after completion of the experiment and seal the exposure module outlet.</li>
	<li>Stop the compressed air supply and the aerosol generator by clicking on the Stop button.</li>
	<li>Open the exposure and clean air module and transfer the cell culture inserts to the prepared post-incubation plates using a tweezer. Incubate the 6-well plates for 24 h (37 &#176;C, 5% CO2) at the ALI. 
	NOTE: Repeat steps 10.5 -10.10 if further exposure experiments are planned.</li>
	<li>Lift the cell culture inserts, that are used as incubator controls to the ALI under the same conditions as the exposed cell culture inserts and incubate them for 24 h (37 &#176;C, 5% CO2) at the ALI.</li>
	<li>Use the button Homing Mode to remove the substance container. Close the aerosol generator software by clicking on the X in the upper right-hand corner and turn off the computer.</li>
	<li>After completion of all exposure experiments, clean the aerosol generator and both exposure modules. Close the substance container with parafilm if the test substance will be further used within the next days.</li>
</ol>
Day 3
11. Cell viability
NOTE: Cell viability was determined 24 h after particle deposition by measuring the mitochondrial activity using the WST-1 assay. The assay was performed according to the manufacturer&#39;s protocol. Cell viability can also be determined by using other cell viability tests (e.g., XTT).
<ol>
	<li>Temper growth medium at 37 &#176;C and thaw the WST-1 solution protected from light. Prepare an appropriate number of new 6-well plates with 2.5 mL growth medium per well and incubate the plates in the incubator.</li>
	<li>Prepare the WST-1 dilution by diluting a sufficient amount of WST-1 1:7 in growth medium</li>
	<li>Insert the cell culture inserts 24 h after exposure in the new prepared 6-well plates. Add 1 mL of the fresh-prepared WST-1 solution to each cell culture insert. Rock the plates carefully in order to distribute the solution homogenously on the cells. Incubate the 6-well plates with the cell culture inserts for 1 h (37 &#176;C, 5% CO2).</li>
	<li>Transfer 100 &#181;L of the supernatant in triplicates from each 6-well to a 96-well plate. Measure the absorbance at 450 nm with a reference wavelength of 650 nm using a microplate reader.</li>
</ol>
12. Statistics
<ol>
	<li>Normalize the cell viability of the individual incubator controls to 100%.</li>
	<li>Express the viability of the exposed cells in relation to the individual incubator controls. Cytotoxicity of test substances was compared to the respective incubator controls and used as an indicator of toxicity.</li>
</ol>

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The authors AT, KG, AB, SH, HM, TG, HT and DS have nothing to disclose. The company Cultex Technology GmbH (formerly Cultex Laboratories GmbH) produces instruments (e.g., CULTEX RFS, CULTEX DG) used in this article. NM was an employee of Cultex Laboratories GmbH during this study. OK is an employee of Cultex Technology GmbH (formerly Cultex Laboratories GmbH). The patent PCT/EP2009/007054 for the device is hold by the founder of the Cultex Technology GmbH Prof. Dr. Ulrich Mohr (formerly Cultex Laboratories GmbH).

Many non-animal inhalation toxicity testing models have been developed in recent years in order to gain information about the acute inhalation hazard of inhalable particles and to reduce and replace animal experiments according to the 3R principle25.
In terms of cell culture models, exposure of cells can be done under submerged conditions or at the ALI. Exposing cells under submerged conditions may affect the physico-chemical properties and thus, the toxic properties of...

Inhalation of toxic airborne particles is a public health concern, leading to a multitude of health risks worldwide and many millions of deaths annually1,2. Climate change, the ongoing industrial development and the rising demand for energy, agricultural and consumer products have contributed to the increase of pulmonary diseases over the last years3,4,5,6. Knowledge and evaluation of inhalable substances regarding their acute inhalation toxicity provide the basis for hazard assessment and risk management, but this information is still lacking for a wide range of these substances7,8. Since 2006, the EU chemical legislation REACH (Registration, Evaluation, Authorization and Restriction of Chemicals) requires that already existing and newly introduced products undergo a toxicological characterization including the inhalation route before being placed on the market. Therefore, REACH focuses on alternative and animal-free methods, the implementation of the &#34;3R&#34; principle (Replacement, Refinement, and Reduction of animal experiments) and the use of appropriate in vitro models9. In recent years, many different and adequate non-animal inhalation toxicity testing models (e.g., in vitro cell cultures, lung-on-a-chip models, precision cut lung slices (PCLS)) have been developed in order to assess the acute inhalation toxicity of airborne particles5,7,10,11. In terms of in vitro cell culture models, cultivated cells can be exposed under submerged conditions or at the ALI (Figure 1). However, the validity of submerged exposure studies is limited with regard to the evaluation of the toxicity of airborne compounds especially particles. Submerged exposure techniques do not correspond to the human in vivo situation; the cell culture medium covering the cells may affect the physico-chemical properties and thus, the toxic properties of a test substance12,13. ALI in vitro inhalation models allow the direct exposure of cells to the test substances without interference of the cell culture medium with test particles, thus, mimicking human exposure with higher physiological and biological similarity than submerged exposures12,14.
For regulatory processes such as REACH, however, only animal models are available in the field of acute inhalation toxicology, as no alternative in vitro methods have been sufficiently validated and officially accepted so far14. For this purpose, test models have to be validated according to the requirements of the European Union Reference Laboratory for Alternatives to Animal Testing (EURL-ECVAM) principles on test validity15.
A former pre-validation study and a recently completed validation study successfully demonstrated the application area of the CULTEX RFS exposure system and its transferability, stability, and reproducibility13. This exposure system is an in vitro cell-based exposure system that enables homogenous exposure of cells to gases, particles or complex atmospheres (e.g., cigarette smoke) at the ALI due to its radial aerosol distribution concept and the conduction of the test aerosol in a continuous flow over the cells16. The basic module of this radial flow system consists of the inlet adapter, the aerosol guiding module with a radial aerosol distribution, the sampling and socket module, and a locking module with a hand wheel (Figure 2). The generated particles reach the cells via the inlet adapter and the aerosol guiding module and are deposited on the cell culture inserts, which are located in the three radially arranged exposure chambers of the sampling module. The aerosol guiding module as well as the sampling module can be heated by connecting to an external water bath17.
Within the framework of both studies, A549 cells were used for all exposure experiments. The cell line A549 is a human immortalized epithelial cell line that is very well-characterized and has been used as an in vitro model for type II alveolar epithelial cells in numerous toxicological studies. The cells are characterized by lamellar bodies, the production of surfactant and a number of inflammation-relevant factors18. They also show properties of bronchial epithelial cells due to their mucus production19. Moreover, they can be cultured at the ALI. Although this cell line is deficient in building cell-cell contacts, the cultivation of these cells is much more convenient, less cost expensive and results derived thereof are donor-independent compared to primary cells20.
A549 cells were seeded in 6-well cell culture inserts (PET membrane, 4.67 cm2, pore size 0.4 mm) with a density of 3.0 x 105 cells per insert and cultivated for 24 h under submerged conditions. Cells were then exposed in three independent laboratories to clean air and three different exposure doses (25, 50, and 100 &#181;g/cm2) of 20 test substances at the ALI. The exposure dose is correlated to the deposition time resulting in a constant particle rate of 25 &#181;g/cm2, 50 &#181;g/cm2 and 100 &#181;g/cm2 onto the cells after 15, 30 or 60 min, respectively. The deposited particles, however, were not washed off after deposition, but remained on the cells for 24 h. The deposition times of the particles were therefore 15, 30 and 60 min, but the exposure of the cells lasted for 24 h in total. The deposition rate of the test substances was determined in preliminary experiments according to previous methods17.
Cell viability as an indicator of toxicity was assessed 24 h after particle deposition using a cell viability assay. Special focus was set on the quality of clean air controls, the optimization and refinement of the exposure protocol, the intra- and inter-laboratory reproducibility and the establishment of a prediction model (PM). Substances that led to a decrease of cell viability below 50% (PM 50%) or 75% (PM 75%) at any of the three exposure doses were considered to exert an acute inhalation hazard. Results were then compared to existing in vivo data (based on at least one reliable study according to OECD test guideline (TG) 403 or TG 43621,22), leading to an overall concordance of 85%, with a specificity of 83% and a sensitivity of 88%23.
Besides the measurement of cell viability, other endpoints such as cytokine release, examination of the cell lysate or membrane integrity via LDH assay can be assessed but were not required for the validation study. Thus, the exposure system (e.g., CULTEX RFS) was proven as a predictive screening system for the qualitative assessment of the acute inhalation toxicity of the tested airborne particles, representing a promising alternative method to animal testing. The following protocol is recommended for exposure experiments to airborne particles using this exposure system.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Cells</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>A549</td>
			<td>ATCC</td>
			<td>CCL-185</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture medium and supplies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Biochrom, Berlin, Germany</td>
			<td>FG 0415</td>
			<td>used as growth medium</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco-Invitrogen, Darmstadt, Germany</td>
			<td>22320</td>
			<td>used as exposure medium</td>
		</tr>
		<tr>
			<td>FBS superior</td>
			<td>Biochrom, Berlin, Germany</td>
			<td>S 0615</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamycin (10mg/mL)</td>
			<td>Biochrom, Berlin, Germany</td>
			<td>A 2710</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES 1M</td>
			<td>Th. Geyer, Renningen, Germany</td>
			<td>L 0180</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Biochrom, Berlin, Germany</td>
			<td>L 1825</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin/EDTA (0.05%/0.02%)</td>
			<td>Biochrom, Berlin, Germany</td>
			<td>L 2143</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture material</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CASY Cups</td>
			<td>Roche Diagnostic GmbH, Mannheim, Germany</td>
			<td>REF 05651794</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture plates</td>
			<td>Corning, Wiesbaden, Germany</td>
			<td>3516</td>
			<td>6&#173;-well plates</td>
		</tr>
		<tr>
			<td>Corning Transwell cell culture inserts</td>
			<td>Corning, Wiesbaden, Germany</td>
			<td>3450</td>
			<td>24mm inserts; 6-&#173;well plates; 0.4 &#181;m</td>
		</tr>
		<tr>
			<td>Chemicals</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CASYton</td>
			<td>Roche Diagnostic GmbH, Mannheim, Germany</td>
			<td>REF 05651808001</td>
			<td></td>
		</tr>
		<tr>
			<td>Compressed Air (DIN EN 12021)</td>
			<td>Linde Gas Therapeutics GmbH, Oberschlei&#223;heim, Germany</td>
			<td>2290152</td>
			<td></td>
		</tr>
		<tr>
			<td>WST-1</td>
			<td>Abcam, Cambridge, United Kingdom</td>
			<td>ab155902</td>
			<td></td>
		</tr>
		<tr>
			<td>Instruments + equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CASY Cell Counter</td>
			<td>Sch&#228;rfe System GmbH, Reutlingen, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Circulation thermostat</td>
			<td>LAUDA, Lauda-K&#246;nigshofen, Germany</td>
			<td>Ecoline RE 100</td>
			<td></td>
		</tr>
		<tr>
			<td>CULTEX HyP - Hydraulic Press</td>
			<td>Cultex&#174; Technology GmbH, Hannover, Gemany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CULTEX insert sleeve</td>
			<td>Cultex&#174; Technology GmbH, Hannover, Gemany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CULTEX RFS - Radial Flow System Type 2 (module for particle exposure)</td>
			<td>Cultex&#174; Technology GmbH, Hannover, Gemany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CULTEX RFS - Radial Flow System Type 2 (module for clean air exposure)</td>
			<td>Cultex&#174; Technology GmbH, Hannover, Gemany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CULTEX supply</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Flow controller 0-30 ml/min (IQ-Flow)</td>
			<td>Bronkhorst Deutschland Nord GmbH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Flow controller 0-1,5 l/min (EL-Flow)</td>
			<td>Bronkhorst Deutschland Nord GmbH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Filters (large)</td>
			<td>Munktell &#38; Filtrak GmbH, Sachsen, Germany</td>
			<td>LP-050</td>
			<td>Munktell Sterile Filter; Particle retention efficiency &#62; 99,999%</td>
		</tr>
		<tr>
			<td>Filters (small)</td>
			<td>Parker Hannifin Corporation, Mainz, Germany</td>
			<td>9933-05-DQ</td>
			<td>Balston disposable filter</td>
		</tr>
		<tr>
			<td>Medium pump</td>
			<td>Cole-Parmer GmbH, Wertheim, Germany</td>
			<td>Ismatec IPC High Precision Multichannel Dispenser</td>
			<td>digital peristaltic pump</td>
		</tr>
		<tr>
			<td>Microplate Reader Infinite M200 Pro</td>
			<td>Tecan Deutschland GmbH, Crailsheim, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Vakuum pump</td>
			<td>KNF, Freiburg, Germany</td>
			<td>N86 KT.18</td>
			<td></td>
		</tr>
		<tr>
			<td>V&#246;gtlin mass flow controller 0,2-10 l/min</td>
			<td>TrigasFI GmbH</td>
			<td>V&#246;gtlin red-y compact regulator, Typ-Nr.: GCR-C3SA-BA20</td>
			<td></td>
		</tr>
		<tr>
			<td>Water Bath</td>
			<td>LAUDA, Lauda-K&#246;nigshofen, Germany</td>
			<td>Ecoline Staredition RE 104</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

The CULTEX RFS is a specially designed modular in vitro exposure system that enables the direct and homogenous exposure of cells at the ALI. Within a former pre-validation study, the general applicability of this exposure system and its transferability, stability and reproducibility were successfully demonstrated. In a recent research project funded by the German Federal Ministry of Education and Research, the exposure system was successfully validated and established as a prediction mode...

Watch this Scientific Journal Video about Assessment of the Acute Inhalation Toxicity of Airborne Particles by Exposing Cultivated Human Lung Cells at the Air-Liquid Interface at JoVE.com

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

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 ...

assessment-acute-inhalation-toxicity-airborne-particles-exposing

Research

JoVE Journal

Medicine

6.9K Views.  Bundeswehr Institute of Pharmacology and Toxicology. 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).

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

Isolation of Mouse Respiratory Epithelial Cells and Exposure to Experimental Cigarette Smoke at Air Liquid Interface

Pulmonary epithelial cells can be isolated from the respiratory tract of mice and cultured at air-liquid interface (ALI) as a model of differentiated respiratory epithelium. A protocol is described for isolating and exposing these cells to mainstream cigarette smoke (CS), in order to study epithelial cell responses to CS exposure. The protocol consists of three parts: the isolation of airway epithelial cells from mouse trachea, the culturing of these cells at air-liquid interface (ALI) as fully differentiated epithelial cells, and the delivery of calibrated mainstream CS to these cells in culture. The ALI culture system allows the culture of respiratory epithelia under conditions that more closely resemble their physiological setting than ordinary liquid culture systems. The study of molecular and lung cellular responses to CS exposure is a critical component of understanding the impact of environmental air pollution on human health. Research findings in this area may ultimately contribute towards understanding the etiology of chronic obstructive pulmonary disease (COPD), and other tobacco-related diseases, which represent major global health problems.

Pulmonary epithelial cells can be isolated from the respiratory tract of mice and cultured at air-liquid interface as a model of differentiated respiratory epithelium. A protocol is described for isolating, culturing and exposing these cells to mainstream cigarette smoke, in order to study molecular responses to this environmental toxin.

Pulmonary epithelial cells can be isolated from the respiratory tract of mice and cultured at air-liquid interface (ALI) as a model of differentiated ...

Isolation of Human Lymphatic Endothelial Cells by Multi-parameter Fluorescence-activated Cell Sorting

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity but little is known about their biology. Isolating highly pure human lymphatic endothelial cells (LECs) from diseased and healthy tissue would facilitate studies of the lymphatic endothelium at genetic, molecular and cellular levels. It is anticipated that these investigations may reveal targets for new therapies that may change the clinical management of these conditions. A protocol describing the isolation of human foreskin LECs and lymphatic malformation lymphatic endothelial cells (LM LECs) is presented. To obtain a single cell suspension tissue was minced and enzymatically treated using dispase II and collagenase II. The resulting single cell suspension was then labelled with antibodies to cluster of differentiation (CD) markers CD34, CD31, Vascular Endothelial Growth Factor-3 (VEGFR-3) and PODOPLANIN. Stained viable cells were sorted on a fluorescently activated cell sorter (FACS) to separate the CD34LowCD31PosVEGFR-3PosPODOPLANINPos LM LEC population from other endothelial and non-endothelial cells. The sorted LM LECs were cultured and expanded on fibronectin-coated flasks for further experimental use.

The goal of this protocol is to isolate lymphatic endothelial cells lining human lymphatic malformation cyst-like vessels and foreskins using fluorescence-activated cell sorting (FACS). Subsequent cell culturing and expansion of these cells permits a new level of experimental sophistication for genetic, proteomic, functional and cell differentiation studies.

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity ...

Diffuse Optical Spectroscopy for the Quantitative Assessment of Acute Ionizing Radiation Induced Skin Toxicity Using a Mouse Model

Acute skin toxicities from ionizing radiation (IR) are a common side effect from therapeutic courses of external beam radiation therapy (RT) and negatively impact patient quality of life and long term survival. Advances in the understanding of the biological pathways associated with normal tissue toxicities have allowed for the development of interventional drugs, however, current response studies are limited by a lack of quantitative metrics for assessing the severity of skin reactions. Here we present a diffuse optical spectroscopic (DOS) approach that provides quantitative optical biomarkers of skin response to radiation. We describe the instrumentation design of the DOS system as well as the inversion algorithm for extracting the optical parameters. Finally, to demonstrate clinical utility, we present representative data from a pre-clinical mouse model of radiation induced erythema and compare the results with a commonly employed visual scoring. The described DOS method offers an objective, high through-put evaluation of skin toxicity via functional response that is translatable to the clinical setting.

We present a diffuse optical spectroscopic (DOS) approach that provides quantitative optical biomarkers of skin response to radiation. We describe DOS instrumentation design, optical parameters extraction algorithms and the animal handling procedures required to yield representative data from a pre-clinical mouse model of radiation induced erythema.

Acute skin toxicities from ionizing radiation (IR) are a common side effect from therapeutic courses of external beam radiation therapy (RT) and ...

Primary Outcome Assessment in a Pig Model of Acute Myocardial Infarction

Mortality after acute myocardial infarction remains substantial and is associated with significant morbidity, like heart failure. Novel therapeutics are therefore required to confine cardiac damage, promote survival and reduce the disease burden of heart failure. Large animal experiments are an essential part in the translational process from experimental to clinical therapies. To optimize clinical translation, robust and representative outcome measures are mandatory. The present manuscript aims to address this need by describing the assessment of three clinically relevant outcome modalities in a pig acute myocardial infarction (AMI) model: infarct size in relation to area at risk (IS/AAR) staining, 3-dimensional transesophageal echocardiography (TEE) and admittance-based pressure-volume (PV) loops. Infarct size is the main determinant driving the transition from AMI to heart failure and can be quantified by IS/AAR staining. Echocardiography is a reliable and robust tool in the assessment of global and regional cardiac function in clinical cardiology. Here, a method for three-dimensional transesophageal echocardiography (3D-TEE) in pigs is provided. Extensive insight into cardiac performance can be obtained by admittance-based pressure-volume (PV) loops, including intrinsic parameters of myocardial function that are pre- and afterload independent. Combined with a clinically feasible experimental study protocol, these outcome measures provide researchers with essential information to determine whether novel therapeutic strategies could yield promising targets for future testing in clinical studies.

Reliable and accurate outcome assessment is the key for translation of preclinical therapies into clinical treatment. The current paper describes how to assess three clinically relevant primary outcome parameters of cardiac performance and damage in a pig acute myocardial infarction model.

Mortality after acute myocardial infarction remains substantial and is associated with significant morbidity, like heart failure. Novel therapeutics are ...

Open Tracheostomy Gastric Acid Aspiration Murine Model of Acute Lung Injury Results in Maximal Acute Nonlethal Lung Injury

Acid pneumonitis is a major cause of sterile acute lung injury (ALI) in humans. Acid pneumonitis spans the clinical spectrum from asymptomatic to acute respiratory distress syndrome (ARDS), characterized by neutrophilic alveolitis, and injury to both alveolar epithelium and vascular endothelium. Clinically, ARDS is defined by acute onset of hypoxemia, bilateral patchy pulmonary infiltrates and non-cardiogenic pulmonary edema. Human studies have provided us with valuable information about the physiological and inflammatory changes in the lung caused by ARDS, which has led to various hypotheses about the underling mechanisms. Unfortunately, difficulties determining the etiology of ARDS, as well as a wide range of pathophysiology have resulted in a lack of critical information that could be useful in developing therapeutic strategies.
Translational animal models are valuable when their pathogenesis and pathophysiology accurately reproduce a concept proven in both in vitro and clinical settings. Although large animal models (e.g., sheep) share characteristics of the anatomy of human trachea-bronchial tree, murine models provide a host of other advantages including: low cost; short reproductive cycle lending itself to greater data acquisition; a well understood immunologic system; and a well characterized genome leading to the availability of a variety of gene deletion and transgenic strains. A robust model of low pH induced ARDS requires a murine ALI that targets mainly the alveolar epithelium, secondarily the vascular endothelium, as well as the small airways leading to the alveoli. Furthermore, a reproducible injury with wide differences between different injurious and non-injurious insults is important.
The murine gastric acid aspiration model presented here using hydrochloric acid employs an open tracheostomy and recreates a pathogenic scenario that reproduces the low pH pneumonitis injury in humans. Additionally, this model can be used to examine interaction of a low pH insult with other pulmonary injurious entities (e.g., food particles, pathogenic bacteria).

This protocol induces acute lung injury in a mouse that has close fidelity to the pathogenesis of acid pneumonitis observed in humans. We generate a maximal acute nonlethal low pH lung injury and account for differences in rodent-human anatomic respiratory structure using an open tracheostomy coupled with circumferential pressure release.

Acid pneumonitis is a major cause of sterile acute lung injury (ALI) in humans. Acid pneumonitis spans the clinical spectrum from asymptomatic to acute ...

Phosphorus-31 Magnetic Resonance Spectroscopy: A Tool for Measuring In Vivo Mitochondrial Oxidative Phosphorylation Capacity in Human Skeletal Muscle

Skeletal muscle mitochondrial oxidative phosphorylation (OXPHOS) capacity, which is critically important in health and disease, can be measured in vivo and noninvasively in humans via phosphorus-31 magnetic resonance spectroscopy (31PMRS). However, the approach has not been widely adopted in translational and clinical research, with variations in methodology and limited guidance from the literature. Increased optimization, standardization, and dissemination of methods for in vivo 31PMRS would facilitate the development of targeted therapies to improve OXPHOS capacity and could ultimately favorably impact cardiovascular health. 31PMRS produces a noninvasive, in vivo measure of OXPHOS capacity in human skeletal muscle, as opposed to alternative measures obtained from explanted and potentially altered mitochondria via muscle biopsy. It relies upon only modest additional instrumentation beyond what is already in place on magnetic resonance scanners available for clinical and translational research at most institutions. In this work, we outline a method to measure in vivo skeletal muscle OXPHOS. The technique is demonstrated using a 1.5 Tesla whole-body MR scanner equipped with the suitable hardware and software for 31PMRS, and we explain a simple and robust protocol for in-magnet resistive exercise to rapidly fatigue the quadriceps muscle. Reproducibility and feasibility are demonstrated in volunteers as well as subjects over a wide range of functional capacities.

Phosphorus-31 Magnetic Resonance Spectroscopy: A Tool for Measuring In Vivo Mitochondrial Oxidative Phosphorylation Capacity in Human Skeletal Muscle

This work demonstrates the feasibility of an in vivo phosphorus-31 magnetic resonance spectroscopy (31PMRS) technique to quantify mitochondrial oxidative phosphorylation (OXPHOS) capacity in human skeletal muscle.

Skeletal muscle mitochondrial oxidative phosphorylation (OXPHOS) capacity, which is critically important in health and disease, can be measured in vivo ...

Inducing Acute Lung Injury in Mice by Direct Intratracheal Lipopolysaccharide Instillation

Airway administration of lipopolysaccharide (LPS) is a common way to study pulmonary inflammation and acute lung injury (ALI) in small animal models. Various approaches have been described, such as the inhalation of aerosolized LPS as well as nasal or intratracheal instillation. The presented protocol describes a detailed step-by-step procedure to induce ALI in mice by direct intratracheal LPS instillation and perform FACS analysis of blood samples, bronchoalveolar lavage (BAL) fluid, and lung tissue. After intraperitoneal sedation, the trachea is exposed and LPS is administered via a 22 G venous catheter. A robust and reproducible inflammatory reaction with leukocyte invasion, upregulation of proinflammatory cytokines, and disruption of the alveolo-capillary barrier is induced within hours to days, depending on the LPS dosage used. Collection of blood samples, BAL fluid, and lung harvesting, as well as the processing for FACS analysis, are described in detail in the protocol. Although the use of the sterile LPS is not suitable to study pharmacologic interventions in infectious diseases, the described approach offers minimal invasiveness, simple handling, and good reproducibility to answer mechanistic immunological questions. Furthermore, dose titration as well as the use of alternative LPS preparations or mouse strains allow modulation of the clinical effects, which can exhibit different degrees of ALI severity or early vs. late onset of disease symptoms.

Presented here is a step-by-step procedure to induce acute lung injury in mice by direct intratracheal lipopolysaccharide instillation and to perform FACS analysis of blood samples, bronchoalveolar lavage fluid, and lung tissue. Minimal invasiveness, simple handling, good reproducibility, and titration of disease severity are advantages of this approach.

Airway administration of lipopolysaccharide (LPS) is a common way to study pulmonary inflammation and acute lung injury (ALI) in small animal models. ...

Oral Health Assessment by Lay Personnel for Older Adults

Oral health is an often-undervalued contributor to overall health. The literature, however, underscores the myriad of systemic diseases influenced by oral health, including type II diabetes, heart disease, and atherosclerosis. Thus, assessments of oral health, called oral screenings, have a significant role in assessing risk of disease, managing disease, and even improving disease by oral care. Here we present a method to assess oral health quickly and consistently across time. The protocol is simple enough for non-oral health professionals such as students, family, and caregivers. Useful for any age of patient, the method is particularly key for older individuals who are often at risk of inflammation and chronic disease. Components of the method include existing oral health assessment scales and inventories, which are combined to produce a comprehensive assessment of oral health. Thus, oral characteristics assessed include intraoral and extraoral structures, soft and hard tissues, natural and artificial teeth, plaque, oral functions such as swallowing, and the impact this oral health status has on the patient&#39;s quality of life. Advantages of this method include its inclusion of measures and perceptions of both the observer and patient, and its ability to track changes in oral health over time. Results acquired are quantitative totals of questionnaire and oral screening items, which can be summed for an oral health status score. The scores of successive oral screenings can be used to track the progression of oral health across time and guide recommendations for both oral and overall health care.

The goal of this protocol is to enable non-dental professionals to assess oral health status for research or health-screening purposes. Aspects assessed include lips, tongue, soft and hard tissues, natural and artificial teeth, oral cleanliness, plaque, swallowing, and impact of oral health on quality of life.

Oral health is an often-undervalued contributor to overall health. The literature, however, underscores the myriad of systemic diseases influenced by oral ...

Employing the Forced Oscillation Technique for the Assessment of Respiratory Mechanics in Adults

There is increasing interest in the use of the forced oscillation technique (FOT) or oscillometry to characterize respiratory mechanics in healthy and diseased individuals. FOT, a complementary method to traditional pulmonary function testing, utilizes a range of oscillatory frequencies superimposed on tidal breathing to measure the functional relationship between airway pressure and flow. This passive assessment provides an estimate of respiratory system resistance (Rrs) and reactance (Xrs) that reflect airway caliber and energy storage and dissipation, respectively. Despite the recent increase in popularity and updated Technical Standards, clinical adoption has been slow which relates, in part, to the lack of standardization regarding the acquisition and reporting of FOT data. The goal of this article is to address the lack of standardization across laboratories by providing a comprehensive written protocol for FOT and an accompanying video. To illustrate that this protocol can be utilized irrespective of a particular device, three separate FOT devices have been employed in the case examples and video demonstration. This effort is intended to standardize the use and interpretation of FOT, provide practical suggestions, as well as highlight future questions that need to be addressed.

As the use of forced oscillation technique (FOT) is increasingly utilized to characterize respiratory mechanics, there is a need to standardize methods with respect to nascent technical guidelines and various manufacturer's recommendations. A detailed protocol is provided including FOT assessment and interpretation for two cases to facilitate the standardization of methods.

There is increasing interest in the use of the forced oscillation technique (FOT) or oscillometry to characterize respiratory mechanics in healthy and ...

Isolation and Profiling of Human Primary Mesenteric Arterial Endothelial Cells at the Transcriptome Level

Endothelial cells (ECs) are crucial for vascular and whole-body function through their dynamic response to environmental cues. Elucidating the transcriptome and epigenome of ECs is paramount to understanding their roles in development, health, and disease, but is limited in the availability of isolated primary cells. Recent technologies have enabled the high-throughput profiling of EC transcriptome and epigenome, leading to the identification of previously unknown EC cell subpopulations and developmental trajectories. While EC cultures are a useful tool in the exploration of EC function and dysfunction, the culture conditions and multiple passages can introduce external variables that alter the properties of native EC, including morphology, epigenetic state, and gene expression program. To overcome this limitation, the present paper demonstrates a method of isolating human primary ECs from donor mesenteric arteries aiming to capture their native state. ECs in the intimal layer are dissociated mechanically and biochemically with the use of particular enzymes. The resultant cells can be directly used for bulk RNA or single-cell RNA-sequencing or plated for culture. In addition, a workflow is described for the preparation of human arterial tissue for spatial transcriptomics, specifically for a commercially available platform, although this method is also suitable for other spatial transcriptome profiling techniques. This methodology can be applied to different vessels collected from a variety of donors in health or disease states to gain insights into EC transcriptional and epigenetic regulation, a pivotal aspect of endothelial cell biology.

The protocol describes the isolation, culture, and profiling of endothelial cells from human mesenteric artery. Additionally, a method is provided to prepare human artery for spatial transcriptomics. Proteomics, transcriptomics, and functional assays can be performed on isolated cells. This protocol can be repurposed for any medium- or large-size artery.