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

Summary

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.

Abstract

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.

Introduction

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.

Protocol

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.......

Discussion

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.......

Materials

Name	Company	Catalog Number	Comments
100x Penicillin/Streptomycin solution	Gibco	15140122
24 mm Transwell with 0.4 µm Pore Polyester Membrane Insert, Sterile	BIOFIL	TCS016012
40 µm Cell Strainer	Falcon	352340
500x Gentamicin/Amphotericin Solution	Gibco	R01510
acetylated α-Tubulin	CST	#5335
Acetyl-α-Tubulin (Lys40) (D20G3)XP Rabbit mAb	cellsignal	#5335
Animal Component Free Cell Dissociation Kit	Stemcell	05426
Anti-pan Cytokeratin antibody	abcam	ab7753
Cigarette	Marlboro
Claudin3	immunoway	YT0949
Deoxyribonuclase I from bovine pancreas	Sigma-Aldrich	DN25
Deoxyribonuclase I from bovine pancreas	Sigma	DN25
Ham’s F-12	Sigma-Aldrich	N6658
Heparin Solution	Stemcell	07980
Hydrocortisone Stock Solution	Stemcell	07925
Mucin 5AC	abcam	ab212636
Occludin	proteintech	27260-1-AP
PBS	Cytosci	CBS004S-BR500
Penicillin-Streptomycin Solution	Gibco	15140122
PneumaCult-ALI Basal Medium	Stemcell	05002
PneumaCult-ALI 10x Supplement	Stemcell	05003
PneumaCult-ALI Maintenance Supplement	Stemcell	05006
PneumaCult-Ex Plus 50x Supplement	Stemcell	05042
PneumaCult-Ex Plus Basal Medium	Stemcell	05041
Pronase E	Sigma-Aldrich	P5147
Rat tail collagen	Corning	354236
Trypan Blue	Stemcell	07050