Name: assessment of the acute inhalation toxicity of airborne particles by exposing cultivated human lung cells at the air-liquid interface
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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), sup...

<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>

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...

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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 ...

The screening of airborne particles concerning the acute pulmonary cytotoxicity by exposing cultivated human lung cells at the ALI using this in vitro exposure system can help to reduce animal experiments. The main advantage of this exposure system is the radial aerosol distribution concept leading to a homogeneous distribution and the position of the particles onto the cells. Before seeding the cells, add 2.5 milliliters of tempered growth medium to each well of a six well plate.Place the cell culture inserts without cells carefully inside the wells and add one milliliter of growth medium to every cell culture insert. Incubate the plates for 30 minutes at 37 degrees Celsius and 5%carbon dioxide. After trypsinization of cells, dilute 100 microliters of the cell culture suspension in a cup filled with 10 milliliters of isotonic solution.Tilt the cup slowly without shaking. On a cell counter, determine the number of viable cells per milliliter and cell viability based on the cell specific measurement parameters of A549 cells. After tempering of the plate, aspirate the medium within the cell culture inserts and seed one milliliter of A549 cells with a density of three times 10 to the fifth cells per milliliters in each cell culture insert.Distribute the cell suspension by gentle rocking and incubate for 24 hours. Set the pressing time via the time control on the front side of the press. Open the compressed air supply at the compressed air valve.Using the pressure regulator on the front side of the press, set the compressed air pressure to approximately two bar. Pull out the drawer, press the Press button, and read the pressing pressure on the digital pressure switch. 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. Place the substance container, with the plunger, in the drawer and press the Press button. Open the drawer and remove the plunger.A critical step is the pressing of test substances that have to be specifically compressed to a powder cake within the substance container in order to enable stable particulate exposures. Remove the inlet adaptor 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.Connect the vacuum lines with the tube connector of the aerosol guiding module. Use the hand wheel to close the module and measure the value of the flow controllers. A couple minutes after closing, the values decrease below five milliliters per minute.Start the aerosol generator software. If the substance scraper is not in the lowest position, press the button Homing Mode. 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. Place the locking plate in the slot over the substance container and tighten the black screw. Change the values for Feed and Rotation to the desired settings.Use the downward arrows to push down the slide with the substance container until the substance scraper is near the pressed substance. 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 to 20 millimeters per hour to avoid long waiting times.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. Start the medium supply with preheated exposure medium and fill the sampling modules until the downpipes are covered while the module is open. Insert blind cell culture inserts 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.Start the aerosol generator, close the exposure module, and connect the exposure module to the exposure module outlet of the aerosol generator. 30 minutes 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 until the downpipes are covered with medium.Remove the cell culture inserts from the six well plates with the help of tweezers. Pour the growth medium carefully from the cell culture inserts off by toppling the inserts and use a pipette to aspirate and discard the residual liquid. Place the inserts in the exposure chambers of both the exposure and clean air modules.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. After completion of the experiment, disconnect the exposure and clean air modules and seal the exposure module outlet. Stop the compressed air supply and the aerosol generator by clicking on the Stop button.Open the exposure and clean air module and use tweezers to transfer the cell culture inserts to the prepared post-incubation six well plates. Incubate the six well plates, as well as the unexposed cell culture inserts, for 24 hours at 37 degrees Celsius and 5%carbon dioxide at the air liquid interface. 24 hours after exposure, insert the cell culture inserts in the new prepared six well plates.Add one milliliter of the fresh prepared WST1 solution to each cell culture insert. Rock the plates carefully in order to distribute the solution homogeneously on the cells. Incubate the six well plates with the cell culture inserts for one hour at 37 degrees Celsius and 5%carbon dioxide.After that, transfer 100 microliters of the supernatant in triplicates from each six well plate to a 96 well plate. Using a microplate reader, measure the absorbance at 450 nanometers with a reference wavelength of 650 nanometers. The CULTEX RFS is a specially designed modular in vitro exposure system that enables the direct and homogenous exposure of cells to airborne particles at the air liquid interface.Exposure of cells to clean air lead to no decrease of cell viability over time. The exposure system was successfully validated and established as a prediction model for acute inhalation hazards of the tested compounds. Exposure of A549 cells to different test substances that exhibited no, medium, or strong toxicity.Please ensure that you strictly keep the time frames that are the incubation times, the lead time of particulate generation, and the particulate deposition times. This exposure method is primarily designed for the particulate exposure of cells, but can be extended to the exposure of liquid aerosols and gasses by adapting the aerosol generation method. This screening method enables the qualitative assessment of inhalable particles regarding the acute inhalation toxicity in vitro, thereby reducing animal testing that would normally provide this toxicological assessment.Don't forget that working with toxic test substances can be extremely hazardous and precautions, such as wearing gloves, a mask, should always be taken when handling such compounds.

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