Name: assessment of the acute inhalation toxicity of airborne particles by exposing cultivated human lung cells at the air-liquid interface
Uploaded: 2020-02-23T14:00:00.000+00:00
Duration: 10 min 10 s
Description: 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

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>

<ol>
	<li>Faber, S. C., McCullough, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Through+the+Looking+Glass:+In+vitro+Models+for+Inhalation+Toxicology+and+Interindividual+Variability+in+the+Airway.">Through the Looking Glass: In vitro Models for Inhalation Toxicology and Interindividual Variability in the Airway.</a> Applied In vitro Toxicology. 4 (2), 115-128 (2018).</li><li>De Matteis, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+and+new+challenges+in+occupational+lung+diseases.">Current and new challenges in occupational lung diseases.</a> European Respiratory Review. 26 (146), 1-15 (2017).</li><li>LANUV Nordrhein-Westfalen. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Gesundheitliche Risiken von Nanomaterialien nach inhalativer Aufnahme. , (2009).</li><li>B&#233;rub&#233;, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+Models+of+Inhalation+Toxicity+and+Disease.+The+report+of+a+FRAME+workshop.">In vitro Models of Inhalation Toxicity and Disease. The report of a FRAME workshop.</a> Alternatives To Laboratory Animals. 37 (1), 89-141 (2009).</li><li>Lopez, A. D., Murray, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+global+burden+of+disease,+1990-2020.">The global burden of disease, 1990-2020.</a> Nature Medicine. 4 (11), 1241-1243 (1998).</li><li>Clippinger, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternative+approaches+for+acute+inhalation+toxicity+testing+to+address+global+regulatory+and+non-regulatory+data+requirements:+An+international+workshop+report.">Alternative approaches for acute inhalation toxicity testing to address global regulatory and non-regulatory data requirements: An international workshop report.</a> Toxicology In vitro. 48, 53-70 (2018).</li><li>Agrawal, M. R., Winder, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Frequency+and+Occurrence+of+LD50+Values+for+Materials+in+the+Workplace.">Frequency and Occurrence of LD50 Values for Materials in the Workplace.</a> Journal Of Applied Toxicology. 16 (5), 407-422 (1996).</li><li>Amtsblatt der Europ&#228;ischen Union. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Verordnung+(EG)+Nr.+1907/2006+des+Europ&#228;ischen+Parlaments+und+des+Rates.">Verordnung (EG) Nr. 1907/2006 des Europ&#228;ischen Parlaments und des Rates.</a> Europ&#228;ische Union. 860, (2006).</li><li>Huh, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstituting+Organ-Level+Lung+Functions+on+a+Chip.">Reconstituting Organ-Level Lung Functions on a Chip.</a> Science. 328 (5986), 1662-1668 (2010).</li><li>Fisher, R. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Use+of+Human+Lung+Slices+in+Toxicology.">The Use of Human Lung Slices in Toxicology.</a> Human and Experimental Toxicology. 13 (7), 466-471 (1994).</li><li>Lenz, A. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammatory+and+Oxidative+Stress+Responses+of+an+Alveolar+Epithelial+Cell+Line+to+Airborne+Zinc+Oxide+Nanoparticles+at+the+Air-Liquid+Interface.">Inflammatory and Oxidative Stress Responses of an Alveolar Epithelial Cell Line to Airborne Zinc Oxide Nanoparticles at the Air-Liquid Interface.</a> Biomed Research International. 12, (2013).</li><li>Steinritz, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+the+CULTEX+Radial+Flow+System+as+an+in+vitro+exposure+method+to+assess+acute+pulmonary+toxicity+of+fine+dusts+and+nanoparticles+with+special+focus+on+the+intra-+and+inter-laboratory+reproducibility.">Use of the CULTEX Radial Flow System as an in vitro exposure method to assess acute pulmonary toxicity of fine dusts and nanoparticles with special focus on the intra- and inter-laboratory reproducibility.</a> Chemico-Biological Interactions. 206 (3), 479-490 (2013).</li><li>Lacroix, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Air-Liquid+Interface+In+vitro+Models+for+Respiratory+Toxicology+Research.">Air-Liquid Interface In vitro Models for Respiratory Toxicology Research.</a> Applied In vitro Toxicology. 4 (2), 91-106 (2018).</li><li>Eskes, C., Whelan, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Validation of Alternative Methods for Toxicity Testing. 418, (2016).</li><li>Rach, J., Budde, J., M&#246;hle, N., Aufderheide, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+exposure+at+the+air-liquid+interface:+Evaluation+of+an+in+vitro+approach+for+simulating+inhalation+of+airborne+substances.">Direct exposure at the air-liquid interface: Evaluation of an in vitro approach for simulating inhalation of airborne substances.</a> Journal Of Applied Toxicology. 34 (5), 506-515 (2014).</li><li>Aufderheide, M., Halter, B., M&#246;hle, N., Hochrainer, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+CULTEX+RFS:+A+comprehensive+Technical+Approach+for+the+In+vitro+Exposure+of+Airway+Epithelial+Cells+to+the+Particulate+Matter+at+the+Air-Liquid+Interface.">The CULTEX RFS: A comprehensive Technical Approach for the In vitro Exposure of Airway Epithelial Cells to the Particulate Matter at the Air-Liquid Interface.</a> Biomed Research International. 15, (2013).</li><li>Lieber, M., Todaro, G., Smith, B., Szakal, A., Nelson-Rees, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+continuous+tumor-cell+line+from+a+human+lung+carcinoma+with+properties+of+type+II+alveolar+epithelial+cells.">A continuous tumor-cell line from a human lung carcinoma with properties of type II alveolar epithelial cells.</a> International Journal Of Cancer. 17 (1), 62-70 (1976).</li><li>Carterson, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A549+lung+epithelial+cells+grown+as+three-dimensional+aggregates:+Alternative+tissue+culture+model+for+Pseudomonas+aeruginosa+pathogenesis.">A549 lung epithelial cells grown as three-dimensional aggregates: Alternative tissue culture model for Pseudomonas aeruginosa pathogenesis.</a> Infection And Immunity. 73 (2), 1129-1140 (2005).</li><li>Kim, K. J., Borok, Z., Crandall, E. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+useful+in+vitro+model+for+transport+studies+of+alveolar+epithelial+barrier.">A useful in vitro model for transport studies of alveolar epithelial barrier.</a> Pharmaceutical Research. 18 (3), 253-255 (2001).</li><li>OECD. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Test+No.+403:+Acute+Inhalation+Toxicity.">Test No. 403: Acute Inhalation Toxicity.</a> OECD Guidelines for the Testing of Chemicals, Section 4. , (2009).</li><li>OECD. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Test+No.+436:+Acute+Inhalation+Toxicity+–+Acute+Toxic+Class+Method.">Test No. 436: Acute Inhalation Toxicity – Acute Toxic Class Method.</a> OECD Guidelines for the Testing of Chemicals, Section 4. , (2009).</li><li>Tsoutsoulopoulos, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Validation+of+the+CULTEX+Radial+Flow+System+for+the+assessment+of+the+acute+inhalation+toxicity+of+airborne+particles.">Validation of the CULTEX Radial Flow System for the assessment of the acute inhalation toxicity of airborne particles.</a> Toxicology In vitro. 58, 245-255 (2019).</li><li>Tsoutsoulopoulos, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+exposure+system+generating+nebulized+aerosol+of+sulfur+mustard+in+comparison+to+the+standard+submerse+exposure.">A novel exposure system generating nebulized aerosol of sulfur mustard in comparison to the standard submerse exposure.</a> Chemico-Biological Interactions. 298, 121-128 (2019).</li><li>Tsoutsoulopoulos, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+the+CULTEX+radial+flow+system+for+in+vitro+investigation+of+lung+damaging+agents.">Optimization of the CULTEX radial flow system for in vitro investigation of lung damaging agents.</a> Toxicology Letters. 244, 28-34 (2016).</li><li>Osman, J. J., Birch, J., Varley, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+response+of+GS-NS0+myeloma+cells+to+pH+shifts+and+pH+perturbations.">The response of GS-NS0 myeloma cells to pH shifts and pH perturbations.</a> Biotechnology and Bioengineering. 75 (1), 63-73 (2001).</li><li>OECD. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Test+Guideline+433:+Acute+Inhalation+Toxicity+–+Acute+Toxic+Class+Method.">Test Guideline 433: Acute Inhalation Toxicity – Acute Toxic Class Method.</a> OECD Guidelines for the Testing of Chemicals, Section 4. , (2018).</li><li>OECD. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Guidance Document on Inhalation Toxicity Studies. , (2018).</li></ol>

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 a test substance12. In vitro ALI inhalation models, however, mimic the human exposure situation with higher biological and physiological similarity than submerged exposure and are therefore better suited for analyzing the acute inhalation toxicity of airborne particles. The significance of the CULTEX RFS with respect to other existing exposure modules is not only the exposure of cells under ALI conditions but also the very homogenous distribution and deposition of particles. In contrast to sequential exposure models with linear aerosol guidance, the modular design of this exposure method enables a radial supply line leading to a very homogenous deposition of particles on the cells17.
The most important point for successful exposure experiments is the stable and congruent quality of the clean air controls. Special attention must be paid that the viability of the clean air controls is not affected over time and as close as possible at 100% compared to the corresponding incubator controls. Factors that play an important role regarding clean air viability are the choice of suitable cell culture inserts, the pH value of the exposure medium, and the composition of the clean air. In terms of cell culture inserts, a good quality and a high density of pores must be guaranteed. This ensures a better medium supply and a higher relative humidity inside the cell culture inserts, protecting the cells from desiccation26. By using cell culture inserts with side wall openings, special insert sleeves must be used in order to avoid leakage of test particles through the side wall openings which could lead to a possibly contamination of the exposure medium. A shift of the pH value higher than 8 can already have a toxic effect on the cells and therefore leading to an impairment of cell viability27. This occurs especially in prolonged particle deposition times (e.g., 60 min) if the clean air contains less than 5% CO2 or the HEPES concentration of the exposure medium is too low which has to be avoided.
A critical issue of the protocol is the pressing of the test substances. The test substances have to be sufficiently compressed to a powder cake within the substance container in order to enable a stable particle exposure. Thus, the substances have to be characterized in preliminary experiments regarding their press properties and in order to obtain information about which press plunger, type of scraping blade or feed rates have to be used.
The maximum pressing pressure of the hydraulic press, however, is 10 kN, which represents at the same time the load limit for the glass cylinder and thus, a limitation of the pressing process. The substance container cannot withstand higher pressing forces than 10 kN. A higher pressing force might offer the pressing of crystalline substances and thus, extend the applicability of this press but would require more robust substance containers.
Moreover, this exposure system which is primarily designed for the investigation of airborne particle exposures 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. Exchanging the aerosol generator with a membrane nebulizer and using a stainless-steel exposure module enabled the exposure of cells to highly toxic liquid aerosols24.
A further critical issue is the overload effect. Cell viability can be affected not only by toxicological properties of a test substance but also by the amount of a substance deposited on the cells. This exposure system shows indeed substantial similarity to the physiological conditions in the human alveolar region but does not contain any clearance mechanisms for removing particles. It is therefore very important that the cell viability is not impaired due to a too large number of particles.
The protocol herein describes the homogenous exposure of cultivated human lung cells at the ALI to airborne particles. The reproducibility, its robustness and transferability make the current exposure system applicable as an in vitro screening method for the qualitative assessment of inhalable particles regarding their acute inhalation toxicity. Because no alternative in vitro methods have been sufficiently validated so far, acute pulmonary toxicity is still being assessed by exposing animals (e.g., whole-body chambers, nose or mouth-only methods). All commonly accepted OECD test guidelines for the acute inhalation toxicity (e.g., TG 403, TG 433 and TG 436) are based on animal models at present21,22,28,29. One future direction will be therefore to apply at the Organization for Economic Cooperation and Development (OECD) for the acceptance as an in vitro test guideline for the acute inhalation toxicity.

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><tbody><tr><td>&lt;strong&gt;Cells&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>A549</td><td>ATCC</td><td>CCL-185</td><td></td></tr><tr><td>&lt;strong&gt;Cell culture medium and supplies&lt;/strong&gt;</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>&lt;strong&gt;Cell culture material&lt;/strong&gt;</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&amp;shy;-well plates</td></tr><tr><td>Corning Transwell cell culture inserts</td><td>Corning, Wiesbaden, Germany</td><td>3450</td><td>24mm inserts; 6-&amp;shy;well plates; 0.4 &amp;micro;m</td></tr><tr><td>&lt;strong&gt;Chemicals&lt;/strong&gt;</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&amp;szlig;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>&lt;strong&gt;Instruments + equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>CASY Cell Counter</td><td>Sch&amp;auml;rfe System GmbH, Reutlingen, Germany</td><td></td><td></td></tr><tr><td>Circulation thermostat</td><td>LAUDA, Lauda-K&amp;ouml;nigshofen, Germany</td><td>Ecoline RE 100</td><td></td></tr><tr><td>CULTEX HyP - Hydraulic Press</td><td>Cultex&amp;reg; Technology GmbH, Hannover, Gemany</td><td></td><td></td></tr><tr><td>CULTEX insert sleeve</td><td>Cultex&amp;reg; 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&amp;reg; 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&amp;reg; 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 &amp;amp; Filtrak GmbH, Sachsen, Germany</td><td>LP-050</td><td>Munktell Sterile Filter; Particle retention efficiency &gt; 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&amp;ouml;gtlin mass flow controller 0,2-10 l/min</td><td>TrigasFI GmbH</td><td>V&amp;ouml;gtlin red-y compact regulator, Typ-Nr.: GCR-C3SA-BA20</td><td></td></tr><tr><td>Water Bath</td><td>LAUDA, Lauda-K&amp;ouml;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 model (PM) for acute inhalation hazards of the tested compounds. As the quality of the clean air controls turned out to be a critical parameter during the pre-validation study, several protocol and method optimizations (e.g., change of cell culture inserts, stabilization of the pH of the exposure medium by increasing the HEPES concentration to 100 mM) were implemented at the beginning of the validation study, leading to highly stable and reproducible results and a substantial improvement of clean air viability data across all three laboratories (Figure 4). A549 cells were then exposed at the ALI to three different exposure doses (25, 50 and 100 &#181;g/cm2) of 20 pre-selected and coded test substances in the three independent laboratories, and cytotoxicity (used as an indicator of toxicity) was compared to the respective incubator controls. Thirteen coded substances were thereby tested in triplicates, seven coded substances as single experiments. Test substances were considered to exert an acute inhalation hazard when cell viability decreased below 50% (PM 50%) or 75% (PM 75%).
As shown exemplarily in Figure 5, exposure of A549 cells to different test substances exhibited no, medium or a strong toxicity. As all experiments were conducted independently in three laboratories, data were analyzed regarding the reproducibility within and between the laboratories and the predictivity of the exposure system. Depending on the applied PM (PM 50% or PM 75%), the within-laboratory and the between-laboratory reproducibility ranged from 90-100%, demonstrating the robustness and transferability of this method. As all tested substances had relevant available in vivo reference data (based on at least one reliable study according to OECD TG 403 or TG 436 using a traditional LC50 protocol and a concentration x time (C x t) protocol), comparison of the in vivo and in vitro data revealed an overall concordance of 85% (17/20) with a specificity of 83% (10/12) and a sensitivity of 85% (7/8) (Table 1). Only two substances were classified as falsely positive and one as falsely negative.
In summary, our results of the validation study present a transferable, reproducible and predictive screening method for the qualitative assessment of the acute pulmonary cytotoxicity of the selected airborne particles.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60572/60572fig1.jpg" /> 
Figure 1: Exposure of cells at the ALI or under submerged conditions. A549 cells can be either exposed with a test substance (blue arrows and dots) at the ALI (left) through an inlet of the exposure system or with the test substance diluted in exposure medium (blue dots) creating submerse conditions (right). Red dotted lines represent the fill levels of exposure medium (bright red) in the respective experimental setup. This figure has been modified from Tsoutsoulopoulos et al.24. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60572/60572fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60572/60572fig2.jpg" /> 
Figure 2: The exposure module. Schematic overview of the basic module of the radial flow system consisting of the inlet adapter, the aerosol guiding module, the sampling and socket module and a locking module with a hand wheel. This figure has been modified from Aufderheide et al.17. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60572/60572fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60572/60572fig3.jpg" /> 
Figure 3: Overview of the exposure system. The components are the two exposure modules for clean air and particle exposure, the aerosol generator including the elutriator and corresponding control unit, and the medium pumps. This figure has been modified from Aufderheide et al.17. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60572/60572fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/60572/60572fig4.jpg" /> 
Figure 4: Clean air viability data after optimization of the test method. Exposure of cells to clean air led to no decrease of cell viability over time, leading to a substantial improvement of clean air viability data compared to the pre-validation study. All clean air controls were pooled for each laboratory (Lab 1-3) and exposure time (n = 46 per laboratory and point in time). Data are displayed as boxplots with a median line and the range indicated by whiskers. This figure has been modified from Tsoutsoulopoulos et al.23. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60572/60572fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/60572/60572fig5.jpg" /> 
Figure 5: Exposure of A549 cells to different test substances. (A) Exposure of A549 cells to tungsten(IV) carbide showed no decrease of cell viability for the three different exposure doses and appeared to be non-toxic (n = 3 per laboratory and exposure dose). (B) Exposure of cells to tetrabromophthalic anhydride resulted for all laboratories in a moderate toxicity presenting a good dose response curve (n = 1 per laboratory and exposure dose). (C) Zinc dimethyldithiocarbamate exhibited a strong toxicity, leading to a decreased cell viability already after a deposited dose of 25 &#181;g/cm2 (n = 3 per laboratory and exposure dose). Error bars represent standard deviations. This figure has been modified from Tsoutsoulopoulos et al.23. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60572/60572fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td colspan="2">Reference results in vivo</td>
			<td colspan="2">Results in vitro</td>
			<td></td>
		</tr>
		<tr>
			<td>Substance</td>
			<td>OECD TG</td>
			<td>CLP regulation</td>
			<td>Toxicity</td>
			<td>PM50%</td>
			<td>PM75%</td>
			<td>Accordance in vivo / in vitro</td>
		</tr>
		<tr>
			<td>Tungsten(IV) carbide</td>
			<td>403</td>
			<td>n. c</td>
			<td>0</td>
			<td>0</td>
			<td>0</td>
			<td>Yes</td>
		</tr>
		<tr>
			<td>Tungsten(IV) carbide nano</td>
			<td>-</td>
			<td>n. c.</td>
			<td>0</td>
			<td>0</td>
			<td>0</td>
			<td>Yes</td>
		</tr>
		<tr>
			<td>N,N&#39;-ethylenebis(N-acetylacetamide)</td>
			<td>EPA OPPTS 870.1300</td>
			<td>n. c.</td>
			<td>0</td>
			<td>0</td>
			<td>0</td>
			<td>Yes</td>
		</tr>
		<tr>
			<td>Silicon dioxide</td>
			<td>403</td>
			<td>n. c.</td>
			<td>0</td>
			<td>0</td>
			<td>0</td>
			<td>Yes</td>
		</tr>
		<tr>
			<td>Diammonium hydrogenortho-phosphate</td>
			<td>403</td>
			<td>n. c.</td>
			<td>0</td>
			<td>0</td>
			<td>0</td>
			<td>Yes</td>
		</tr>
		<tr>
			<td>Disodium fluorophosphate</td>
			<td>403</td>
			<td>n. c.</td>
			<td>0</td>
			<td>0</td>
			<td>0</td>
			<td>Yes</td>
		</tr>
		<tr>
			<td>Neodymium oxide</td>
			<td>436</td>
			<td>n. c.</td>
			<td>0</td>
			<td>0</td>
			<td>0</td>
			<td>Yes</td>
		</tr>
		<tr>
			<td>Potassium hydrogen monopersulfate</td>
			<td>403</td>
			<td>n. c.</td>
			<td>0</td>
			<td>0</td>
			<td>0</td>
			<td>Yes</td>
		</tr>
		<tr>
			<td>Cyclohepta-pentylose</td>
			<td>403</td>
			<td>n. c.</td>
			<td>0</td>
			<td>0</td>
			<td>0(2x) / 1(1x)</td>
			<td>(Yes)</td>
		</tr>
		<tr>
			<td>Vanadium(III) oxide</td>
			<td>403</td>
			<td>n. c.</td>
			<td>0</td>
			<td>0</td>
			<td>0(2x) / 1(1x)</td>
			<td>(Yes)</td>
		</tr>
		<tr>
			<td>Tetrapotassium pyrophosphate</td>
			<td>403</td>
			<td>n. c.</td>
			<td>0</td>
			<td>1</td>
			<td>1</td>
			<td>No</td>
		</tr>
		<tr>
			<td>Tetra-bromophthalic anhydride</td>
			<td>403 (similar)</td>
			<td>n. c.</td>
			<td>0</td>
			<td>1</td>
			<td>1</td>
			<td>No</td>
		</tr>
		<tr>
			<td>Cetylpyridinium chloride</td>
			<td>403</td>
			<td>Acute Tox. 2</td>
			<td>1</td>
			<td>1</td>
			<td>1</td>
			<td>Yes</td>
		</tr>
		<tr>
			<td>N-Lauroylsarcosine sodium salt</td>
			<td>403</td>
			<td>Acute Tox. 2</td>
			<td>1</td>
			<td>1</td>
			<td>1</td>
			<td>Yes</td>
		</tr>
		<tr>
			<td>Zinc dimethyldithio-carbamate</td>
			<td>403</td>
			<td>Acute Tox. 2</td>
			<td>1</td>
			<td>1</td>
			<td>1</td>
			<td>Yes</td>
		</tr>
		<tr>
			<td>Copper(II) hydroxide</td>
			<td>403</td>
			<td>Acute Tox. 2</td>
			<td>1</td>
			<td>1</td>
			<td>1</td>
			<td>Yes</td>
		</tr>
		<tr>
			<td>Zinc selenite</td>
			<td>436</td>
			<td>Acute Tox. 3</td>
			<td>1</td>
			<td>1</td>
			<td>1</td>
			<td>Yes</td>
		</tr>
		<tr>
			<td>Sodium metavanadate</td>
			<td>403</td>
			<td>Acute Tox. 4</td>
			<td>1</td>
			<td>1</td>
			<td>1</td>
			<td>Yes</td>
		</tr>
		<tr>
			<td>Divanadium pentaoxide</td>
			<td>403</td>
			<td>Acute Tox. 4</td>
			<td>1</td>
			<td>1</td>
			<td>1</td>
			<td>Yes</td>
		</tr>
		<tr>
			<td>Cadmium telluride</td>
			<td>403</td>
			<td>Acute Tox. 4 (n. c.)</td>
			<td>1</td>
			<td>0</td>
			<td>0</td>
			<td>No</td>
		</tr>
		<tr>
			<td colspan="7">n. c. = not classified; 0 = non-toxic; 1 = toxic; CLP = classification, labelling and packaging</td>
		</tr>
	</tbody>
</table>
Table 1: Accordance between in vivo and in vitro results. Out of 20 substances, 10 substances were classified as correctly negative, and seven substances correctly as positive, leading to a concordance of 85% (17/20). This table has been modified from Tsoutsoulopoulos et al.23. (Test No. 403: Acute Inhalation Toxicity, Test No. 436: Acute Inhalation Toxicity - Acute Toxic Class Method; Acute Tox. 2 = fatal, Acute Tox. 3 = toxic, Acute Tox. 4 = harmful).
<img alt="Supplementary Figure 1" src="/files/ftp_upload/60572/60572suppfig1b.jpg" /> 
Supplementary Figure 1: Assembly of the substance container. Picture taken from CULTEX DG (Dust Generator) User Manual. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60572/60572suppfig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Supplementary Figure 2" src="/files/ftp_upload/60572/60572suppfig2.jpg" /> 
Supplementary Figure 2: Aerosol guiding module of the exposure system. A) Top view and B) bottom view of the aerosol guiding module. Picture taken from CULTEX RFS (Radial Flow System) User Manual. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60572/60572suppfig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Supplementary Figure 3" src="/files/ftp_upload/60572/60572suppfig3.jpg" /> 
Supplementary Figure 3: The aerosol generator. A) Schematic overview of the aerosol generator, consisting of the aerosol generator top and the Elutriator. Detailed view of B) the aerosol generator top and C) the Elutriator. Picture taken from CULTEX DG (Dust Generator) User Manual. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60572/60572suppfig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Supplementary Figure 4" src="/files/ftp_upload/60572/60572suppfig4.jpg" /> 
Supplementary Figure 4: The aerosol generator control software. Picture taken from CULTEX DG (Dust Generator) User Manual. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60572/60572suppfig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

Nanyang Technological University

Research

JoVE Journal

Medicine

6.9K Views.  Bundeswehr Institute of Pharmacology and Toxicology. Het screenen van deeltjes in de lucht met betrekking tot de acute longcytotoxiciteit door het blootstellen van gecultiveerde menselijke longcellen in het ALI met behulp van dit in vitro blootstellingssysteem kan helpen om dierproeven te verminderen. Het belangrijkste voordeel van dit blootstellingssysteem is het radiale aerosol distributieconcept dat leidt tot een homogene verdeling en de positie van de deeltjes op de cellen. Voordat u de cellen zaait, voegt u 2,5 milliliter getemperd groeime...

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.

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 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 ...

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.

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.

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

Environment

In vitro Cell Culture Model for Toxic Inhaled Chemical Testing

Cell cultures are indispensable to develop and study efficacy of therapeutic agents, prior to their use in animal models. We have the unique ability to model well differentiated human airway epithelium and heart muscle cells. This could be an invaluable tool to study the deleterious effects of toxic inhaled chemicals, such as chlorine, that can normally interact with the cell surfaces, and form various byproducts upon reacting with water, and limiting their effects in submerged cultures. Our model using well differentiated human airway epithelial cell cultures at air-liqiuid interface circumvents this limitation as well as provides an opportunity to evaluate critical mechanisms of toxicity of potential poisonous inhaled chemicals. We describe enhanced loss of membrane integrity, caspase release and death upon toxic inhaled chemical such as chlorine exposure. In this article, we propose methods to model chlorine exposure in mammalian heart and airway epithelial cells in culture and simple tests to evaluate its effect on these cell types.

In vitro Cell Culture Model for Toxic Inhaled Chemical Testing

This protocol is designed to demonstrate exposure method of cell cultures to inhaled toxic chemicals. Exposure of differentiated air-liquid&#160;interface (ALI) cultures of airway epithelial cells provides a unique model of airway exposure to toxic gases such as chlorine. In this manuscript we describe effect of chlorine exposure on air-liquid&#160;interface cultures of epithelial cells and submerged culture of cardiomyocytes. In vitro exposure systems allow important mechanistic studies to evaluate pathways that could then be utilized to develop novel therapeutic agents.

Cell cultures are indispensable to develop and study efficacy of therapeutic agents, prior to their use in animal models. We have the unique ability to ...

Bioengineering

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 ...

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

Generating 3D Spheres and 2D Air-Liquid Interface Cultures of Human Induced Pluripotent Stem Cell-Derived Type 2 Alveolar Epithelial Cells

In the lung, the alveolar epithelium is a physical barrier from environmental stimuli and plays an essential role in homeostasis and disease. Type 2 alveolar epithelial cells (AT2s) are the facultative progenitors of the distal lung epithelium. Dysfunction and injury of AT2s can result from and contribute to various lung diseases. Improved understanding of AT2 biology is, thus, critical for understanding lung biology and disease; however, primary human AT2s are generally difficult to isolate and limited in supply. To overcome these limitations, human induced pluripotent stem cell (iPSC)-derived type 2 alveolar epithelial cells (iAT2s) can be generated through a directed differentiation protocol that recapitulates in vivo lung development. iAT2s grow in feeder-free conditions, share a transcriptomic program with human adult primary AT2s, and execute key functions of AT2s such as production, packaging, and secretion of surfactant. This protocol details the methods for maintaining self-renewing iAT2s through serial passaging in three-dimensional (3D) culture or adapting iAT2s to air-liquid interface (ALI) culture. A single-cell suspension of iAT2s is generated before plating in 3D solubilized basement membrane matrix (hereafter referred to as &#34;matrix&#34;), where they self-assemble into monolayered epithelial spheres. iAT2s in 3D culture can be serially dissociated into single-cell suspensions to be passaged or plated in 2D ALI culture. In ALI culture, iAT2s form a polarized monolayer with the apical surface exposed to air, making this platform readily amenable to environmental exposures. Hence, this protocol generates an inexhaustible supply of iAT2s, producing upwards of 1 x 1030 cells per input cell over 15 passages while maintaining the AT2 program indicated by SFTPCtdTomato expression. The resulting cells represent a reproducible and relevant platform that can be applied to study genetic mutations, model environmental exposures, or screen drugs.

The present protocol describes human induced pluripotent stem cell-derived type 2 alveolar epithelial-like cells (iAT2s). These cells can be cultured as self-renewing spheres in 3D culture or adapted to air-liquid interface (ALI) culture.

In the lung, the alveolar epithelium is a physical barrier from environmental stimuli and plays an essential role in homeostasis and disease. Type 2 ...

Developmental Biology

Assessment of the Cytotoxic and Immunomodulatory Effects of Substances in Human Precision-cut Lung Slices

Respiratory diseases in their broad diversity need appropriate model systems to understand the underlying mechanisms and enable development of new therapeutics. Additionally, registration of new substances requires appropriate risk assessment with adequate testing systems to avoid the risk of individuals being harmed, for example, in the working environment. Such risk assessments are usually conducted in animal studies. In view of the 3Rs principle and public skepticism against animal experiments, human alternative methods, such as precision-cut lung slices (PCLS), have been evolving. The present paper describes the ex vivo technique of human PCLS to study the immunomodulatory potential of low-molecular-weight substances, such as ammonium hexachloroplatinate (HClPt). Measured endpoints include viability and local respiratory inflammation, marked by altered secretion of cytokines and chemokines. Pro-inflammatory cytokines, tumor necrosis factor alpha (TNF-&#945;), and interleukin 1 alpha (IL-1&#945;) were significantly increased in human PCLS after exposure to a sub-toxic concentration of HClPt. Even though the technique of PCLS has been substantially optimized over the past decades, its applicability for the testing of immunomodulation is still in development. Therefore, the results presented here are preliminary, even though they show the potential of human PCLS as a valuable tool in respiratory research.

In view of the 3Rs principle, respiratory models as alternatives to animal studies are evolving. Especially for risk assessment of respiratory substances, there is a lack of appropriate assays. Here, we describe the use of human precision-cut lung slices for the assessment of airborne substances.

Respiratory diseases in their broad diversity need appropriate model systems to understand the underlying mechanisms and enable development of new ...

Immunology and Infection

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

Visualizing Lung Cellular Adaptations during Combined Ozone and LPS Induced Murine Acute Lung Injury

Lungs are continually faced with direct and indirect insults in the form of sterile (particles or reactive toxins) and infectious (bacterial, viral or fungal) inflammatory conditions. An overwhelming host response may result in compromised respiration and acute lung injury, which is characterized by lung neutrophil recruitment as a result of the patho-logical host immune, coagulative and tissue remodeling response. Sensitive microscopic methods to visualize and quantify murine lung cellular adaptations, in response to low-dose (0.05 ppm) ozone, a potent environmental pollutant in combination with bacterial lipopolysaccharide, a TLR4 agonist, are crucial in order to understand the host inflammatory and repair mechanisms. We describe a comprehensive fluorescent microscopic analysis of various lung and systemic body compartments, namely the broncho-alveolar lavage fluid, lung vascular perfusate, left lung cryosections, and sternal bone marrow perfusate. We show damage of alveolar macrophages, neutrophils, lung parenchymal tissue, as well as bone marrow cells in correlation with a delayed (up to 36-72 h) immune response that is marked by discrete chemokine gradients in the analyzed compartments. In addition, we present lung extracellular matrix and cellular cytoskeletal interactions (actin, tubulin), mitochondrial and reactive oxygen species, anti-coagulative plasminogen, its anti-angiogenic peptide fragment angiostatin, the mitochondrial ATP synthase complex V subunits, &#945; and &#946;. These surrogate markers, when supplemented with adequate in vitro cell-based assays and in vivo animal imaging techniques such as intravital microscopy, can provide vital information towards understanding the lung response to novel immunomodulatory agents.

Combined ozone and bacterial endotoxin exposed mice show wide-spread cell death, including that of neutrophils. We observed cellular adaptations such as disruption of cytoskeletal lamellipodia, increased cellular expression of complex V ATP synthase subunit &#946; and angiostatin in broncho-alveolar lavage, suppression of the lung immune response and delayed neutrophil recruitment.

Lungs are continually faced with direct and indirect insults in the form of sterile (particles or reactive toxins) and infectious (bacterial, viral or ...

Multicellular Human Alveolar Model Composed of Epithelial Cells and Primary Immune Cells for Hazard Assessment

A human alveolar cell coculture model is described here for simulation of the alveolar epithelial tissue barrier composed of alveolar epithelial type II cells and two types of immune cells (i.e., human monocyte-derived macrophages [MDMs] and dendritic cells [MDDCs]). A protocol for assembling the multicellular model is provided. Alveolar epithelial cells (A549 cell line) are grown and differentiated under submerged conditions on permeable inserts in two-chamber wells, then combined with differentiated MDMs and MDDCs. Finally, the cells are exposed to an air-liquid interface for several days. As human primary immune cells need to be isolated from human buffy coats, immune cells differentiated from either fresh or thawed monocytes are compared in order to tailor the method based on experimental needs. The three-dimensional models, composed of alveolar cells with either freshly isolated or thawed monocyte-derived immune cells, show a statistically significant increase in cytokine (interleukins 6 and 8) release upon exposure to proinflammatory stimuli (lipopolysaccharide and tumor necrosis factor &#945;) compared to untreated cells. On the other hand, there is no statistically significant difference between the cytokine release observed in the cocultures. This shows that the presented model is responsive to proinflammatory stimuli in the presence of MDMs and MDDCs differentiated from fresh or thawed peripheral blood monocytes (PBMs). Thus, it is a powerful tool for investigations of acute biological response to different substances, including aerosolized drugs or nanomaterials.

Presented here is a protocol for primary human blood monocyte isolation as well as their differentiation into macrophages and dendritic cells and assembly with epithelial cells into a multicellular human lung model. Biological responses of cocultures composed of immune cells differentiated from either freshly isolated or thawed monocytes, upon exposure to proinflammatory stimuli, are compared.

A human alveolar cell coculture model is described here for simulation of the alveolar epithelial tissue barrier composed of alveolar epithelial type II ...

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

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