This study was supported by the National Natural Science Foundation of China (82090013).

The overall protocol requires 44 days, including 1 day for preparation of airway epithelial cells isolated from murine tracheas, 15 days for cell proliferation, and 28 days for CSE stimulation at the air-liquid interface. All experimental animals are housed in the SPF Barrier Animal Room of the Animal Experiment Center of Capital Medical University and have been reviewed and approved by the Animal Experiment and Laboratory Animal Ethics Committee of Capital Medical University (AEEI-2020-100) to meet the requirements of A...

In Vitro Model of Murine Airway Epithelial Cells and Chronic Smoke Exposure

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COPD is a common chronic airway inflammatory disease. Exposure to tobacco smoke leads to chronic airway inflammation, airway remodeling, and lung structural destruction, which is the result of the interaction of various structural cells and immune cells10. As the front line of the innate immune system in the lung, airway epithelial cells play a very important role during the development of the disease11. In this point of view, clarifying how epithelial cells change and regu...

Chronic obstructive pulmonary disease (COPD) is a heterogeneous lung condition with complex characteristics, while patients with COPD gradually tend to be younger1. Smoking, a primary risk factor for COPD2, has a profound impact on airway epithelial cells, which serve as the initial barrier against tobacco smoke. Despite this known association, the detailed mechanisms through which tobacco smoke induces changes in airway epithelial cells remain inadequately explored. A thorough understanding of these molecular alterations is essential for identifying early diagnostic markers and therapeutic targets for COPD.
To address this gap, we developed a novel in vitro model using murine airway epithelial cells. These cells were subjected to long-term stimulation with cigarette smoke extract (CSE), enabling us to monitor dynamic cellular changes and explore the changes in airway epithelial cells under long-term tobacco stimulation and the underlying mechanism. In this model, transwell was used to provide the air-liquid interface of airway epithelial cells, and cigarette smoke extract was stimulated in the early stage of epithelial cell differentiation until the end of differentiation at 28 days. Previous studies investigated only the differentiation and short-term stimulation of airway epithelial cells (cell line dominance). They were limited to a single regulatory pathway3,4,5,6. However, the protocol presented here uses primary mouse airway epithelial cells and optimizes the cell extraction process for better cell activity than previous airway epithelial cell culture methods. This model focuses on alterations in cellular differentiation alongside comprehensive transcriptomic, proteomic, metabolomic, and epigenomic analyses. Employing immunofluorescence and advanced multi-omics techniques, we aimed to elucidate the cellular responses of airway epithelial cells to chronic tobacco smoke exposure, thereby contributing to a deeper understanding of COPD pathogenesis. This model can be used to explore the changes in airway epithelial cell differentiation pattern and its mechanism caused by long-term stimulation of various pollutants.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>100x Penicillin/Streptomycin solution</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>24 mm Transwell with 0.4 &#181;m Pore Polyester Membrane Insert, Sterile</td>
			<td>BIOFIL</td>
			<td>TCS016012</td>
			<td></td>
		</tr>
		<tr>
			<td>40 &#181;m Cell Strainer</td>
			<td>Falcon</td>
			<td>352340</td>
			<td></td>
		</tr>
		<tr>
			<td>500x Gentamicin/Amphotericin Solution</td>
			<td>Gibco</td>
			<td>R01510</td>
			<td></td>
		</tr>
		<tr>
			<td>acetylated &#945;-Tubulin</td>
			<td>CST</td>
			<td>#5335</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetyl-&#945;-Tubulin (Lys40) (D20G3)XP Rabbit mAb&#160;</td>
			<td>cellsignal</td>
			<td>#5335</td>
			<td></td>
		</tr>
		<tr>
			<td>Animal Component Free Cell Dissociation Kit</td>
			<td>Stemcell</td>
			<td>05426</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-pan Cytokeratin antibody</td>
			<td>abcam</td>
			<td>ab7753</td>
			<td></td>
		</tr>
		<tr>
			<td>Cigarette</td>
			<td>Marlboro</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Claudin3</td>
			<td>immunoway</td>
			<td>YT0949</td>
			<td></td>
		</tr>
		<tr>
			<td>Deoxyribonuclase I from bovine pancreas</td>
			<td>Sigma-Aldrich</td>
			<td>DN25</td>
			<td></td>
		</tr>
		<tr>
			<td>Deoxyribonuclase I from bovine pancreas</td>
			<td>Sigma</td>
			<td>DN25</td>
			<td></td>
		</tr>
		<tr>
			<td>Ham&#8217;s F-12</td>
			<td>Sigma-Aldrich</td>
			<td>N6658</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin Solution&#160;</td>
			<td>Stemcell</td>
			<td>07980</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrocortisone Stock Solution</td>
			<td>Stemcell</td>
			<td>07925</td>
			<td></td>
		</tr>
		<tr>
			<td>Mucin 5AC</td>
			<td>abcam</td>
			<td>ab212636</td>
			<td></td>
		</tr>
		<tr>
			<td>Occludin</td>
			<td>proteintech</td>
			<td>27260-1-AP</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Cytosci</td>
			<td>CBS004S-BR500</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin Solution</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>PneumaCult-ALI 
			Basal Medium</td>
			<td>Stemcell</td>
			<td>05002&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>PneumaCult-ALI 10x Supplement</td>
			<td>Stemcell</td>
			<td>05003&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>PneumaCult-ALI Maintenance Supplement</td>
			<td>Stemcell</td>
			<td>05006</td>
			<td></td>
		</tr>
		<tr>
			<td>PneumaCult-Ex Plus 50x Supplement</td>
			<td>Stemcell</td>
			<td>05042</td>
			<td></td>
		</tr>
		<tr>
			<td>PneumaCult-Ex Plus Basal Medium</td>
			<td>Stemcell</td>
			<td>05041</td>
			<td></td>
		</tr>
		<tr>
			<td>Pronase E</td>
			<td>Sigma-Aldrich</td>
			<td>P5147</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat tail collagen</td>
			<td>Corning</td>
			<td>354236</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue</td>
			<td>Stemcell</td>
			<td>07050&#160;</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vitro model for studying differentiation and changes of multi-omics on murine airway epithelial cells stimulated with cigarette smoke extract

Chronic obstructive pulmonary disease (COPD) is largely attributed to tobacco smoke exposure. Investigating how airway epithelial cells functionally adapt to tobacco smoke is crucial for understanding the pathogenesis of COPD. The present study was to set up an in vitro model using primary murine airway epithelial cells to mimic the real-life impact of tobacco smoke. Unlike established cell lines, primary cells retain more in vivo-like properties, including growth patterns, aging, and differentiation. These cells exhibit a sensitive inflammatory response and efficient differentiation, thus closely representing physiological conditions. In this model, primary murine airway epithelial cells were cultured for 28 days under an air-liquid interface with an optimal concentration of cigarette smoke extract (CSE), which led to the transformation of a monolayer of undifferentiated cells into a pseudostratified columnar epithelium, indicative of CSE acclimation. Comprehensive multi-omics analyses were then applied to elucidate the mechanisms by which CSE influences the differentiation of basal airway cells. These insights provide a deeper understanding of the cellular processes underpinning COPD progression in response to tobacco smoke exposure.

Author Spotlight: Development and Characterization of an In Vitro Model to Study Chronic Cigarette Smoke Exposure and Its Impact on Airway Epithelial Cells in COPD Research

In Vitro Model for Studying Differentiation and Changes of Multi-Omics on Murine Airway Epithelial Cells Stimulated with Cigarette Smoke Extract

The scope of our research is to develop and characterize an in vitro model using airway epithelial cells to study the effects of chronic CSC exposure. We aimed to understand the cellular response that occurred in airway epithelial cells under long-term cigarette smoke exposure, which is relevant to the pathogenesis of COPD. The recognition of intricate epithelium immune cell interaction in COPD is considered a pivotal recent development in COPD research.We have established a novel in vitro model that mimic chronic CSC exposure in airway epithelial cells. Our findings open up new avenue of investigating the long-term effect of chronic cigarette smoke exposure on airway epithelial cells and their role in COPD progression. In the future, our laboratory will focus on exploring specific pathway that regulate epithelial cells differentiation and the role of airway epithelial cells heterogenetic in COPD treatment response.

Differentiation 
Murine airway epithelial cells successfully differentiated after culturing at an air-liquid interface with a differentiation medium for 28 days. The presence of ciliated and goblet cells was demonstrated by immunofluorescence assay of cilia marker acetylated &#945;-Tubulin (green; Figure 3A) and the goblet cell marker Mucin5AC, respectively6 (red; Figure 3B).
Determination of CSE concentr...

Here, we describe an in vitro model for isolating and differentiating murine airway epithelial cells, focusing on their acclimation to chronic cigarette smoke extract (CSE). The model could be utilized to comprehensively characterize the multi-omics impact of CSE, which possibly provides insights into the cellular responses under chronic smoke exposure.

Chronic obstructive pulmonary disease (COPD) is largely attributed to tobacco smoke exposure. Investigating how airway epithelial cells functionally adapt ...