The authors would like to thank Maurice Smith and Marja Talikka for their review of the manuscript.

<ol>
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This study was funded by Philip Morris International.

Here, we have demonstrated the applicability of human organotypic bronchial and nasal tissue models to assess the impact of repeated CS exposure. As an alternative to animal testing, a number of exposure systems were developed for toxicological assessments of aerosol exposure in vitro (e.g., Vitrocell, Cultex, ALICE, etc.). These exposure modules can also be utilized for a toxicological assessment of airborne pollutants, airborne particles, nanoparticles, etc. In this study, we used the Vitrocell system that can accomodate up to 48 different samples simultaneously, allowing for larger scale experiments and lower variability between treatments. For every aerosol exposure in vitro, the risk of tissue culture contamination remains a major risk whose mitigation demands a careful handling of the tissue cultures throughout the experiments.
Measuring particle deposition in real time on the microbalance during the exposure experiment allows to monitor that the CS doses generated from the exposure system are conforming to the expectations. To ensure the accuracy of the measured particle deposition, setting-up the online measurement correctly before the exposure is critial, e.g., setting up the scale to 0. Additionally, because of the difference between the areas of the microbalance (cm2) and the tissue culture insert (0.33 cm2), we adjusted the final calculation to the area of the culture insert: only 33% of the deposition on the microbalance reflect the actual deposition in the culture insert.
Measurement of TEER to ascertain tight-junction barrier functionality and to assess disruption of the epithelial layer is a relatively easy procedure to implement, which we reported here. However, because the bronchial and nasal tissue models contain mucus-producing interspersed goblet cells, apical washing needs to be performed before the TEER measurement. The apical washing is critical because the presence of the mucus layer and the variability of its thickness can bias the measurement of TEER, interferring with the impact of CS exposure. This notion is in agreement with what was reported by Hilgendorf and colleagues, in which the permeability of Caco-2 cells was affected by the co-culturing with the mucus-producing goblet cell line HT29 23. The mucus needs to be washed off prior to TEER measurement, because apical washing right before the exposure may interfere with the tissue responses to CS. Therefore, the measurement is performed three days before exposure, and not right before exposure.
We showed that CYP1A1/CYP1B1 activity could be measured from the organotypic culture models following CS exposure although the activity was only modestly increased by CS. This weak signal can be amplified by a longer incubation of the CYP1A1/1B1 substrate (i.e., luciferin-CEE), for example for 24 hr (data not shown). One of the limitations of the CYP activity measurement in the present work is the absence of normalization to the CYP protein level or to the cell count, which could be taken into consideration for future studies to ensure that the alteration on the enzyme activity is not influenced by the alteration of either the protein level or the cell count.
We reported that CS exposure inhibited the ciliary beating in both the nasal and bronchial tissue models. Similar observations were done in diverse mammalian and non-mammalian models 12. For ciliary beating measurement, ensuring that the tissues are handled and treated in a similar manner is critical, for example if medium change is implemented, it should be applied for all samples. Sutto and colleagues reported that pH affected the mammalian ciliary beating frequency 24. Thus, when comparing beating frequencies between cells treated with different compunds/mixtures, pH adjustment should be considered to minimize the variability of ciliary beating measurement. Moreover, the temperature at which the measurement is conducted is also critical as the frequency of ciliary beating drops with decreasing temperature. To minimize the variability due to these changes, a stage-top incubator, equiped with temperature, CO2, and humidity control was used in this study. Despite this, we observed that the ciliary beating frequencies in the bronchial tissues after CS exposure were highly variable (i.e., increase in one insert and decrease in the other insert), suggesting that the ciliary beating is highly disturbed right after exposure. In contrast, we observed the absence of measurable ciliary beating frequencies in the nasal tissue after CS exposure, suggesting that the response of the nasal tissue is more sensitive and consistent. This is in agreement with a previous publication showing that the nasal tissue has a lower capacity to detoxify as compared with the bronchus 25.
Finally, we showed that gene expression profiling from the organotypic bronchial and nasal tissue models exposed to CS could demonstrate an impact of CS on xenobiotic metabolism. Interestingly, the observed alteration in xenobiotic metabolism in the organotypic bronchial and nasal in vitro models exposed to CS resemble that of the in vivo situation in smokers as discussed in greater detail in a previous publication 15. For gene expression analyses, using the robotic instrument makes a high-throughput analysis possible. Moreover, automatic robotic handling further increases the consistency and accuracy of the gene expression results. Nonetheless, fast collection of the tissue samples was critical to avoid RNA degradation during the RNA extraction. The RNA processing and transcriptomic approaches described here can also be applied to in vivo tissue samples.

Lungs are directly and constantly exposed to inhaled air that may contain diverse toxicants such as pollutants and constituents of cigarette smoke (CS). Studying the impact of exposure to those toxicants on respiratory tissues is most informative when done in a manner that resembles in vivo exposure. Compared with the classical 2D immersed cell cultures (e.g., normal human bronchial epithelial cells (NHBE)), 3D organotypic tissue models better recapitulate the morphological, physiological, and molecular aspects of the human airway epithelium in vivo1,2: the 3D tissue models contain the diversity of the cell types observed in vivo, including differentiated epithelial cells, ciliated and non-ciliated cells, goblet cells, and basal cells. They have functional tight junctions and exhibit a mucociliary phenotype 1-3. Moreover, the cultures can be grown on a permeable porous membrane, in an air-liquid interface, allowing a direct exposure to aerosol at the apical side (whereas the basolateral side is immersed in culture medium) 3-5. Dvorak and colleagues reported that gene expression profiles of bronchial tissue models were similar to those obtained from human bronchial brushings 3. In addition, Mathis and colleagues showed that the responses of these tissue models to CS were similar to the differences observed between bronchial epithelial cells obtained from smokers and cells obtained from non-smokers 6. Finally, because the bronchial tissue models could be cultured for up to several months 4,5, they could potentially be used to examine the effects of long-term exposure of test items. 
Cytotoxicity assessments are common parameters measured following chemical insults or to assess the toxicity of specific compounds or mixtures. For instance, membrane integrity can be measured by a luminescent assay and allows the measurement of a dose-dependent cytotoxic effect on the cell culture 7. However, to assess pathophysiological effects of compounds at subtoxic concentrations, other parameters should be measured. For example, tissue integrity determined using the transepithelial electrical resistance (TEER) assay ensures the functionality of tight junctions and monitors the disruption of the epithelial layer 8,9. Ciliary beating frequency also allows the measurement of CS-related effects on respiratory tissues. A normal beating frequency for the cilia lining bordering the upper and lower respiratory tract is important to protect against airway infections 10. Each of the ciliated columnar epithelial cells of the respiratory epithelium has 200-300 cilia beating at a particular frequency to eliminate infectious agents or inhaled particulate matter trapped in the mucus released by interspersed goblet cells 11. CS contains chemicals that may inhibit ciliary beating 12, leading to a reduced protection of the respiratory tract. This work shows that ciliary beating can be measured in organotypic tissue models. This approach allows assessment of whether epithelial cells exhibit their normal function in the organotypic tissue culture. CS also activates xenobiotic metabolism responses in the respiratory tract to metabolize tobacco smoke constituents 13. The activity of the phase I xenobiotic metabolism enzymes, CYP1A1 and CYP1B1, of the tissue models can be measured. Additionally, as previously reported, global gene expression can be measured in the organotypic bronchial tissue models 6,14,15. A transcriptomic data and network-based systems biology approach is leveraged to assess the impact of CS on xenobiotic metabolism 15. 
The methodologies used to expose organotypic 3D bronchial and nasal tissue models to mainstream CS using an in vitro exposure system and to measure the tissue responses to this exposure compared to fresh air exposure (control) are detailed here.

<table border="0" cellpadding="0" cellspacing="0" width="1545">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:356px;">Name of Material/ Equipment</td>
			<td style="width:367px;">Company</td>
			<td style="width:655px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MucilAir-human fibroblasts-bronchia</td>
			<td>Epithelix S&#225;rl, Geneva, Switzerland</td>
			<td>http://www.epithelix.com/content/view/122/19/lang,en/</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MucilAir Culture Medium</td>
			<td>Epithelix S&#225;rl, Geneva, Switzerland</td>
			<td>http://www.epithelix.com/content/view/84/16/lang,en/</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">VITROCELL</td>
			<td>VITROCELL systems GmbH, Waldkirch, Germany</td>
			<td>http://www.vitrocell.com/index.php?Nav_Nummer=2&#38;R=</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3R4F reference cigarette</td>
			<td>University of Kentucky</td>
			<td>http://www2.ca.uky.edu/refcig/</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">30-port carousel smoking machine SM2000</td>
			<td>Philip Morris, Int.</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CiliaMetrix camera and software</td>
			<td>La Haute &#201;cole de Gestion (HESGE), Geneva, Switzlerland</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Leica DMIL microscope&#160;</td>
			<td>Leica, Heerbrugg, Switzerland</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LED light source</td>
			<td>Titan Tool Supplies, Buffalo, NY</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Chopstick Electrode STX-2</td>
			<td>World Precision Instruments</td>
			<td>http://www.wpiinc.com/products/physiology/stx2-chopstick-electrode-set-for-evom2/</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EVOMXTM Epithelial Voltohmmeter&#160;</td>
			<td>World Precision Instruments</td>
			<td>http://www.wpiinc.com/products/physiology/evom2-evom2-epithelial-voltohmmeter-for-teer/</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Luciferin Detection Reagent&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MagNA Lyser Instrument&#160;</td>
			<td>Roche</td>
			<td>http://www.roche.com/products/product-details.htm?region=us&#38;type=product&#38;id=66</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">chloroform&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>http://www.sigmaaldrich.com/catalog/product/sial/288306?lang=en&#38;region=</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">QIAcube&#160;</td>
			<td>Qiagen</td>
			<td align="right">9001882</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NanoDrop</td>
			<td>Thermo Scientific</td>
			<td>http://www.nanodrop.com/</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agilent 2100 Bioanalyzer&#160;</td>
			<td>Agilent</td>
			<td>http://www.genomics.agilent.com/en/Bioanalyzer-System/2100-Bioanalyzer-Instruments/?cid=AG-PT-106</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Affymetrix GeneChip High throughput 3&#8217;IVT Express Kit</td>
			<td>Affymetrix</td>
			<td>http://www.affymetrix.com/catalog/prod370001/AFFY/High-Throughput-(HT)-Whole-Transcript-(WT)-Kit</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Scanner 3000 7G&#160;</td>
			<td>Affymetrix</td>
			<td>http://www.affymetrix.com/catalog/131503/AFFY/Scanner-3000-7G</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

In results described below, the organotypic tissues were primary human epithelial cells isolated from healthy, non-smoking, Caucasian donors and were reconstituted using fibroblasts 15. 
&#160;
3D bronchial and nasal tissue models 
In vitro bronchial and nasal organotypic models resemble at the cellular level the human respiratory tract epithelium (Figure 2). 
&#160;
CS exposure 
Organotypic bronchial and nasal tissue models can be repeatedly exposed to mainstream CS at the air-liquid interface. The smoke exposure experimental procedure used for the results illustrated here is depicted in Figure 1. 
&#160;
Recording of ciliary beating 
Cilia beating was recorded in the air-exposed tissues as illustrated in the videos. CS exposure was associated with a reduction of the ciliary beating frequency: the stronger peak observed around 4 Hz in the sham exposed and before exposure is not observed any longer after CS exposure (Figure 3). This reduced frequency would make the cilia beating less efficient to remove mucus and infectious agents 22. 
&#160;
Measurement of TEER and CYP1A1/1B1 activity 
TEER was measured 48 hr after the last exposure to ensure the tissue is still functional. CYP1A1/1B1 activity was also measured 48 h after end of exposure as illustrated in Figure 1. The TEER values in the CS-exposed tissues were not significantly different as compared to the air-exposed tissues confirming that there is no major cytotoxic effect of smoke at this concentration. At the 48 h post-exposure, CYP1A1/1B1 activity of the tissues was slightly increased, indicating a modest increase of tissue defense mechanism following the exposure (Figure 4). 
&#160;
Gene expression profiles and network-based systems biology approach to study the impact of CS exposure on the alteration of xenobiotic metabolism 
Tissues were collected at 48 h post-exposure time points. Subsequently, microarray analysis was conducted to generate gene expression profiles of the CS- and air-exposed tissues. Impact of the CS exposure on xenobiotic metabolism was investigated using a network-based systems biology approach leveraged from the gene-expression profiles and as reported earlier 15. Using a network-based systems biology approach shows that CS exposure impacts at the level of backbone nodes in the Xenobiotic Metabolism network model were nicely correlated between the in vitro and in vivo datasets (Figure 5). In contrast, at the level of each individual gene-expression changes, poor correlations were observed between the in vitro (CS-exposed vs. air-exposed) and in vivo (smokers vs. nonsmokers).
<img alt="Figure 1" src="/files/ftp_upload/52325/52325fig1.jpg" /> 
Figure 1. Schematic procedure of the repeated exposure of CS to the organotypic bronchial tissue models. Abbreviations: CYP, cytochrome P450s; CS, cigarette smoke; TEER, transepithelial electrical resistance.
<img alt="Figure 2" src="/files/ftp_upload/52325/52325fig2.jpg" /> 
Figure 2. Organotypic bronchial and nasal epithelial tissue culture models. Cartoon illustrating the bronchial (A) and nasal (B) tissue culture insert at the air-liquid interface. The in vitro models contained ciliated cells shown in the apical layer of the Hematoxylin &#38; Eosin stained cells (C for bronchial, D for nasal) as reported previously 15. The models were co-cultured with fibroblasts that are important for the growth and differentiation of epithelial cells (indicated by arrows). Staining of airway lineage markers: p63 (E for bronchial, F for nasal) and Muc5AC (G for bronchial, H for nasal) are shown as reported previously 15. <a href="https://www.jove.com/files/ftp_upload/52325/52325fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/52325/52325fig3.jpg" /> 
Figure 3.&#160;Cilia Beating Frequency. Cilia beating frequency (CBF) was measured in air-exposed and in the culture before and after exposure. Decreased CBF was seen after CS exposure (representative results from two insert are shown as replicate 1 (R1) and replicate 2 (R2)).
<img alt="Figure 4" src="/files/ftp_upload/52325/52325fig4.jpg" /> 
Figure 4. Measurement of CYP1A1/CYP1B1 activity and TEER. Left pannels indicate the CYP1A1/CYP1B1 activity in the bronchial (A) and nasal (B) tissue models. Right panels indicate the TEER measurement in the bronchial (C) and nasal (D) tissue models. Means &#177; SD are shown. Abbreviations: CS, cigarette smoke; CYP, cytochrome P450; TEER, transepithelial electrical resistance; RLU, relative luminescense unit.
<img alt="Figure 5" src="/files/ftp_upload/52325/52325fig5.jpg" /> 
Figure 5. Network-based systems biology approach for the impact assessment of CS exposure in the context of xenobiotic metabolism. The changes of gene expressions levels were used to compute the activation of backbone node of the Xenobiotic Metabolism network model (A) as reported in a previous publication 15. The figure on the right illustrates the concept of the quantification of the backbone nodes, also known as &#8220;differential network backbone value,&#8221; using the NPA approach. The blue ovals represent the activity of the backbone nodes (i.e., the functional layer) and the green balls represent the expression of genes (i.e., the transcriptional layer). Impacts of CS exposure in organotypic bronchial tissues (in vitro, CS-exposed vs. air-exposed) at the 48 h of postexposure were compared to those of smoking on human bronchial epithelial collected by bronchoscopy and nasal epithelial by burshing the inferior turbinate (GSE16008) (in vivo, smokers vs. nonsmokers) (B). Insets, correlation of the gene expression changes between the in vitro and in vivo datasets. Abbreviations: CS, cigarette smoke; NPA, network perturbation amplitude. <a href="https://www.jove.com/files/ftp_upload/52325/52325fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Impact Assessment of Repeated Exposure of Organotypic 3D Bronchial and Nasal Tissue Culture Models to Whole Cigarette Smoke at JoVE.com

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

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 ...

impact-assessment-repeated-exposure-organotypic-3d-bronchial-nasal

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Bioengineering

11.0K Görüntüleme.  Philip Morris Products S.A.. 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.