Name: in vitro model for studying differentiation and changes of multi-omics on murine airway epithelial cells stimulated with cigarette smoke extract
Uploaded: 2024-07-12T14:50:00.000+00:00
Duration: 9 min 7 s
Description: Chronic obstructive pulmonary disease (COPD) is largely attributed to tobacco smoke exposure. Investigating how airway epithelial cells functionally adapt ...

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 ARRIVE guidelines7.
1. Isolation of primary murine airway epithelial cells from murine tracheas
<ol>
	<li>Preparation
	<ol>
		<li>Prepare complete expansion medium by combining 1 mL of 50x supplement and 0.05 mL of hydrocortisone stock with 48.95 mL expansion basal medium. Store at 4 &#176;C and use within 4 weeks.</li>
		<li>Dissolve 7.5 mg of proteinase and 5 mg of deoxyribonuclease I in 5 mL of Ham&#39;s F-12 to prepare a proteinase solution. Filter it to sterilize and store at 4 &#176;C and use within 4 weeks.</li>
		<li>Prepare antibiotic solutions by adding 0.05 mL of 100x penicillin/streptomycin and 0.01 mL of 500x gentamicin/amphotericin to 5 mL of complete expansion medium, PBS, and proteinase solution (100 U/mL Penicillin, 100 U/mL Streptomycin, 10 &#181;g/mL gentamicin and 0.25 &#181;g/mL amphotericin B). Store at 4 &#176;C and use within 4 weeks.</li>
		<li>Sterilize surgical instruments and prepare the biological safety cabinet for cell isolation. Simultaneously, ensure that standard cell culture equipment is ready for use.</li>
		<li>Prepare rat tail collagen-coated dishes by adding rat tail collagen (100 &#181;g/mL in 0.02 N acetic acid) into 100 mm culture dishes and 24 mm transwells (0.05 mL/cm2). Leave open overnight under UV light for sterilization.</li>
	</ol>
	</li>
	<li>Isolation of primary murine airway epithelial cells
	<ol>
		<li>Use C57BL/6 mice (6-8 weeks old, male, SPF-raised) for tracheal epithelial cell isolation. Euthanize with 1% pentobarbital sodium solution overdose (about 200 mg/kg). Use five mice for sufficient cell yield.</li>
		<li>Sterilize the mouse by immersion in 75% ethanol solution, not immersing the nose and mouth in alcohol to prevent alcohol from flowing into the trachea.</li>
		<li>Dissect the mouse.
		<ol>
			<li>Use surgical blades and forceps, one set of devices for cutting and opening the epidermis from the lower jaw to the abdominal cavity of the mouse.</li>
			<li>Use another set of surgical instruments to tear apart the thyroid glands on both sides and remove the connections between the trachea, the surrounding muscle tissue, and the esophagus.</li>
			<li>After that, carefully cut into the chest, use forceps to delve deeper into the chest cavity, take out the entire lung, and find the end of the trachea.</li>
		</ol>
		</li>
		<li>Process the trachea.
		<ol>
			<li>Cut out the trachea from the thyroid cartilage to the tracheo-bronchial branch and put it in a pre-cold expansion medium containing four antibiotics.</li>
			<li>Shake the tube to rinse as much blood as possible off the surface of the trachea, keep it on ice, and then start the surgery for the next mouse.</li>
		</ol>
		</li>
		<li>Transfer all collected tracheas to pre-cold PBS buffer containing four antibiotics (200 U/mL Penicillin, 200 U/mL Streptomycin, 20 &#181;g/mL gentamicin, and 0.5 &#181;g/mL amphotericin B) for pretreatment before digestion.</li>
		<li>Use a set of surgical instruments to remove clots and other tissue from the surfaces of the trachea, and then cut them into 1 cm2 size.</li>
		<li>Incubate tracheal tissue in proteinase solution at 37 &#176;C for 40 min.</li>
		<li>After 40 min, rock the tube several times and use a 40 &#181;m cell strainer to remove the remaining tissues. Rinse the chopped tracheas on a strainer with 5 mL of expansion medium, centrifuge the cell suspension at 400 x g for 5 min at 4 &#176;C, and discard the supernatant.</li>
		<li>Resuspend the obtained cells in 8 mL of expansion medium.</li>
		<li>Count the viable cells using trypan blue and a hemocytometer.</li>
		<li>Plate P0 cell suspension on 100 mm dishes (1 x 104 live cells/cm2) precoated with rat tail collagen, and culture the cells in an incubator at 37 &#176;C, 5% CO2.</li>
	</ol>
	</li>
</ol>
2. Expansion culture and passage of primary murine airway epithelial cells
<ol>
	<li>Replace the medium for the P0 cell culture every 3 days until cells reach 70%-80% confluency, usually within 6 days. At this point, murine tracheal epithelial cells can form an intact monolayer with a cobblestone appearance (Figure 1A-D).</li>
	<li>Pre-warm sufficient volumes of PBS, complete expansion medium, and animal component-free (ACF) cell dissociation kit to 37 &#176;C.</li>
	<li>Gently rinse the cells with 3 mL of PBS.</li>
	<li>Perform enzymatic dissociation.
	<ol>
		<li>Add 3 mL of ACF enzymatic dissociation solution to the dish and incubate at 37 &#176;C for 5 min. After pipetting, dislodge the cells gently and transfer the cell suspension to 3 mL of ACF enzyme inhibition solution.</li>
		<li>Repeat the addition of 3 mL of ACF enzymatic dissociation solution and incubate again for 5 min to ensure maximum cell detachment.</li>
	</ol>
	</li>
	<li>Spin the cell suspension at 400 x g for 5 min at 4 &#176;C. Discard the supernatant.</li>
	<li>Resuspend the cells in 1 mL of expansion medium.</li>
	<li>Count the viable cells using trypan blue and a hemocytometer.</li>
	<li>Plate P1 cell suspension on several 100 mm dishes (5 x 104 live cells/cm2) pre-coated with rat tail collagen, and culture the cells in an incubator at 37 &#176;C, 5% CO2.</li>
</ol>
3. Differentiation and CSE stimulation of primary murine airway epithelial cells at air-liquid interface
<ol>
	<li>Confirm the cell type.
	<ol>
		<li>Verify the epithelial origin of isolated cells using an immunofluorescence assay (IFA) with antibodies against cytokeratins. Seed cells on glass slides coated by rat tail collagen and use paraformaldehyde to fix them for at least 12 h after cells reach 80% confluency.</li>
		<li>Wash slides 3 times with PBS solution for 5 min and incubate them with 3% BSA and 0.1% triton X-100 in PBS solution at room temperature (RT) for 1 h.</li>
		<li>Incubate slides overnight at 4 &#176;C with commercial anti-pan cytokeratin antibody at a dilution of 1:500.</li>
		<li>The next day, wash slides 3 times with PBS solution for 5 min and incubate them with secondary antibody buffer at a dilution of 1:1000 at RT for 2 h in the dark.</li>
		<li>After incubation, wash slides 3 times with PBS (5 min per wash), and add DAPI at a dilution of 1:1000 to them. After incubation at RT for 15 min, wash slides once with PBS for 5 min.</li>
		<li>Observe slides under a fluorescence microscope and compare expression patterns to a positive control (A549 cell line) and a negative control (Raw264.7 cell line) (Figure 2).</li>
	</ol>
	</li>
	<li>Seed the cells for differentiation.
	<ol>
		<li>Detach and centrifuge P2 cells as previously described, and resuspend the cell pellet in an appropriate volume of expansion medium to facilitate plating of 1 x 104 live cells/cm2.</li>
		<li>Pipette 1 mL of cell suspension onto the apical chamber of the transwell polycarbonate membrane insert. To the basal compartment of the transwell, add 1.5 mL of proliferation media.</li>
	</ol>
	</li>
	<li>Incubate the cells at 37 &#176;C and fully change all medium in the basal and apical chambers every 3 days using expansion medium until confluence is reached. This typically takes 3-6 days.</li>
	<li>Prepare 50 mL of complete differentiation medium by adding 5 mL of ALI 10x Supplement, 500 &#181;L of ALI Maintenance Supplement, 100 &#181;L of heparin solution, and 250 &#181;L of hydrocortisone stock solution to 44.15 mL of ALI Basal Medium.</li>
	<li>Prepare CSE.
	<ol>
		<li>Bubble one cigarette through 12.5 mL of differentiation medium and then filter it through a 0.22 &#181;m pore filter to prepare CSE. To ensure standardization between experiments and batches of CSE, measure the absorbance at 320 nm on a spectrophotometer and define optical density (OD) of 1 as 100%8.</li>
	</ol>
	</li>
	<li>Use the differentiation medium containing an appropriate concentration of CSE to stimulate primary murine airway epithelial cells to detect effects on the differentiation of the cells.</li>
	<li>Allow cells to differentiate for 28 days, during which the basal chamber media is changed, and the apical side is washed twice a week by removing the apical chamber media and replacing the basal chamber media with the differentiation medium containing the appropriate concentration of CSE.</li>
	<li>Analyze the cells post-exposure.
	<ol>
		<li>After exposure to differentiation medium containing CSE, harvest the cells and the culture supernatants at any time point (i.e., 0-28 days) for detecting morphological changes (i.e., immunofluorescence assay (IFA) or hematoxylin-eosin staining (HE) or for bioinformatics analytical procedures (i.e., transcriptomics, proteomics, metabolomics, etc.).</li>
	</ol>
	</li>
</ol>

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

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The authors have nothing to disclose.

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><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 &amp;micro;m Pore Polyester Membrane Insert, Sterile</td><td>BIOFIL</td><td>TCS016012</td><td></td></tr><tr><td>40 &amp;micro;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 &amp;alpha;-Tubulin</td><td>CST</td><td>#5335</td><td></td></tr><tr><td>Acetyl-&amp;alpha;-Tubulin (Lys40) (D20G3)XP Rabbit mAb&amp;nbsp;</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&amp;rsquo;s F-12</td><td>Sigma-Aldrich</td><td>N6658</td><td></td></tr><tr><td>Heparin Solution&amp;nbsp;</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&lt;br/&gt; Basal Medium</td><td>Stemcell</td><td>05002&amp;nbsp;</td><td></td></tr><tr><td>PneumaCult-ALI 10x Supplement</td><td>Stemcell</td><td>05003&amp;nbsp;</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&amp;nbsp;</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.

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...

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

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.

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 ...

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Biology

896 Views.  Capital Medical University. De reikwijdte van ons onderzoek is het ontwikkelen en karakteriseren van een in vitro model met behulp van luchtwegepitheelcellen om de effecten van chronische blootstelling aan CSC te bestuderen. We wilden de cellulaire respons begrijpen die optrad in luchtwegepitheelcellen onder langdurige blootstelling aan sigarettenrook, wat relevant is voor de pathogenese van COPD. De herkenning van ingewikkelde interactie tussen epitheel en immuuncellen bij COPD wordt b...

Video: Auteur in de schijnwerpers: ontwikkeling en karakterisering van een in vitro model om chronische blootstelling aan sigarettenrook en de impact ervan op luchtwegepitheelcellen te bestuderen in COPD-

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 ...

Medicine

Optimization of the Wound Scratch Assay to Detect Changes in Murine Mesenchymal Stromal Cell Migration After Damage by Soluble Cigarette Smoke Extract

Cell migration is vital to many physiological and pathological processes including tissue development, repair, and regeneration, cancer metastasis, and inflammatory responses. Given the current interest in the role of mesenchymal stromal cells in mediating tissue repair, we are interested in quantifying the migratory capacity of these cells, and understanding how migratory capacity may be altered after damage. Optimization of a rigorously quantitative migration assay that is both easy to customize and cost-effective to perform is key to answering questions concerning alterations in cell migration in response to various stimuli. Current methods for quantifying cell migration, including scratch assays, trans-well migration assays (Boyden chambers), micropillar arrays, and cell exclusion zone assays, possess a range of limitations in reproducibility, customizability, quantification, and cost-effectiveness. Despite its prominent use, the scratch assay is confounded by issues with reproducibility related to damage of the cell microenvironment, impediments to cell migration, influence of neighboring senescent cells, and cell proliferation, as well as lack of rigorous quantification. The optimized scratch assay described here demonstrates robust outcomes, quantifiable and image-based analysis capabilities, cost-effectiveness, and adaptability to other applications.

The capacity to migrate is a key function of many different cell types, including mesenchymal stromal cells (MSCs). However, quantifying alterations in migratory capacity after damage is challenging. This protocol describes an easily adaptable migration assay that uses rigorous statistics to quantify changes in MSC migratory capacity after damage.

Cell migration is vital to many physiological and pathological processes including tissue development, repair, and regeneration, cancer metastasis, and ...

Developmental Biology

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 ...

Bioengineering

Generation of ESC-derived Mouse Airway Epithelial Cells Using Decellularized Lung Scaffolds

Lung lineage differentiation requires integration of complex environmental cues that include growth factor signaling, cell-cell interactions and cell-matrix interactions. Due to this complexity, recapitulation of lung development in vitro to promote differentiation of stem cells to lung epithelial cells has been challenging. In this protocol, decellularized lung scaffolds are used to mimic the 3-dimensional environment of the lung and generate stem cell-derived airway epithelial cells. Mouse embryonic stem cell are first differentiated to the endoderm lineage using an embryoid body (EB) culture method with activin A. Endoderm cells are then seeded onto decellularized scaffolds and cultured at air-liquid interface for up to 21 days. This technique promotes differentiation of seeded cells to functional airway epithelial cells (ciliated cells, club cells, and basal cells) without additional growth factor supplementation. This culture setup is defined, serum-free, inexpensive, and reproducible. Although there is limited contamination from non-lung endoderm lineages in culture, this protocol only generates airway epithelial populations and does not give rise to alveolar epithelial cells. Airway epithelia generated with this protocol can be used to study cell-matrix interactions during lung organogenesis and for disease modeling or drug-discovery platforms of airway-related pathologies such as cystic fibrosis.

This protocol efficiently directs mouse embryonic stem cell-derived definitive endoderm to mature airway epithelial cells. This differentiation technique uses 3-dimensional decellularized lung scaffolds to direct lung lineage specification, in a defined, serum-free culture setting.

Lung lineage differentiation requires integration of complex environmental cues that include growth factor signaling, cell-cell interactions and ...

Automated Measurement of Pulmonary Emphysema and Small Airway Remodeling in Cigarette Smoke-exposed Mice

COPD is projected to be the third most common cause of mortality world-wide by 2020(1). Animal models of COPD are used to identify molecules that contribute to the disease process and to test the efficacy of novel therapies for COPD. Researchers use a number of models of COPD employing different species including rodents, guinea-pigs, rabbits, and dogs(2). However, the most widely-used model is that in which mice are exposed to cigarette smoke. Mice are an especially useful species in which to model COPD because their genome can readily be manipulated to generate animals that are either deficient in, or over-express individual proteins. Studies of gene-targeted mice that have been exposed to cigarette smoke have provided valuable information about the contributions of individual molecules to different lung pathologies in COPD(3-5). Most studies have focused on pathways involved in emphysema development which contributes to the airflow obstruction that is characteristic of COPD. However, small airway fibrosis also contributes significantly to airflow obstruction in human COPD patients(6), but much less is known about the pathogenesis of this lesion in smoke-exposed animals. To address this knowledge gap, this protocol quantifies both emphysema development and small airway fibrosis in smoke-exposed mice. This protocol exposes mice to CS using a whole-body exposure technique, then measures respiratory mechanics in the mice, inflates the lungs of mice to a standard pressure, and fixes the lungs in formalin. The researcher then stains the lung sections with either Gill&#8217;s stain to measure the mean alveolar chord length (as a readout of emphysema severity) or Masson&#8217;s trichrome stain to measure deposition of extracellular matrix (ECM) proteins around small airways (as a readout of small airway fibrosis). Studies of the effects of molecular pathways on both of these lung pathologies will lead to a better understanding of the pathogenesis of COPD.

The goal of this protocol is to provide automated methods to quantify chronic lung pathologies in a murine model of COPD. The protocol includes exposing mice to cigarette smoke (CS), measuring pulmonary function, inflating the lungs, and using morphometry methods to measure emphysema and small airway remodeling in mice.

COPD is projected to be the third most common cause of mortality world-wide by 2020(1).  Animal models of COPD are used to identify molecules that ...

Airway Model for Studying Lung Barrier Integrity, Disease, and Drug Discovery

iPSC-Derived Epithelial, Mesenchymal, Endothelial, and Immune Cell Co-Culture to Model Airway Barrier Integrity in Lung Health and Disease

Human lung tissue is composed of an interconnected network of epithelium, mesenchyme, endothelium, and immune cells from the upper airway of the nasopharynx to the smallest alveolar sac. Interactions between these cells are crucial in lung development and disease, acting as a barrier against harmful chemicals and pathogens. Current in vitro co-culture models utilize immortalized cell lines with different biological backgrounds, which may not accurately represent the cellular milieu or interactions of the lung. We differentiated human iPSCs into 3D lung organoids (containing both epithelium and mesenchyme), endothelial cells, and macrophages. These were co-cultured in an air-liquid interface (ALI) format to form an epithelial/mesenchymal apical barrier invested with macrophages and a basolateral endothelial barrier (iAirway). iPSC-derived iAirways showed a reduction in barrier integrity in response to infection with respiratory viruses and cigarette toxins. This multi-lineage lung co-culture system provides a platform for studying cellular interactions, signaling pathways, and molecular mechanisms underlying lung development, homeostasis, and disease progression. iAirways closely mimic human physiology and cellular interactions, can be generated from patient-derived iPSC&#39;s, and can be customized to include different cell types of the airway. Overall, iPSC-derived iAirway models offer a versatile and powerful tool for studying barrier integrity to better understand genetic drivers for disease, pathogen response, immune regulation, and drug discovery or repurposing in vitro, with the potential to advance our understanding and treatment of airway diseases.

Author Spotlight: Modeling Human Airway Remodeling and Viral Responses Using Isogenic Epithelial, Endothelial, and Immune Cells

This article describes the generation of a complex, multi-cellular airway barrier model composed of induced pluripotent stem cell (iPSC)-derived lung epithelium, mesenchyme, endothelial cells, and macrophages in an air-liquid interface culture.

Human lung tissue is composed of an interconnected network of epithelium, mesenchyme, endothelium, and immune cells from the upper airway of the ...

Studying Effects of Cigarette Smoke on Pseudomonas Infection in Lung Epithelial Cells

Cigarette smoking is the major etiological cause for lung emphysema and chronic obstructive pulmonary disease (COPD). Cigarette smoking also promotes susceptibility to bacterial infections in the respiratory system. However, the effects of cigarette smoking on bacterial infections in human lung epithelial cells have yet to be thoroughly studied. Described here is a detailed protocol for the preparation of cigarette smoking extracts (CSE), treatment of human lung epithelial cells with CSE, and bacterial infection and infection determination. CSE was prepared with a conventional method. Lung epithelial cells were treated with 4% CSE for 3 h. CSE-treated cells were, then, infected with Pseudomonas at a multiplicity of infection (MOI) of 10. Bacterial loads of the cells were determined by three different methods. The results showed that CSE increased Pseudomonas load in lung epithelial cells. This protocol, therefore, provides a simple and reproducible approach to study the effect of cigarette smoke on bacterial infections in lung epithelial cells.

Studying Effects of Cigarette Smoke on Pseudomonas Infection in Lung Epithelial Cells

Described here is a protocol to study how cigarette smoke extract affects bacterial colonization in lung epithelial cells.

Cigarette smoking is the major etiological cause for lung emphysema and chronic obstructive pulmonary disease (COPD). Cigarette smoking also promotes ...

Immunology and Infection

Isolating Bronchial Epithelial Cells from Resected Lung Tissue for Biobanking and Establishing Well-Differentiated Air-Liquid Interface Cultures

The airway epithelial cell layer forms the first barrier between lung tissue and the outside environment and is thereby constantly exposed to inhaled substances, including infectious agents and air pollutants. The airway epithelial layer plays a central role in a large variety of acute and chronic lung diseases, and various treatments targeting this epithelium are administered by inhalation. Understanding the role of epithelium in pathogenesis and how it can be targeted for therapy requires robust and representative models. In vitro epithelial culture models are increasingly being used and offer the advantage of performing experiments in a controlled environment, exposing the cells to different kinds of stimuli, toxicants, or infectious agents. The use of primary cells instead of immortalized or tumor cell lines has the advantage that these cells differentiate in culture to a pseudostratified polarized epithelial cell layer with a better representation of the epithelium compared to cell lines.
Presented here is a robust protocol, that has been optimized over the past decades, for the isolation and culture of airway epithelial cells from lung tissue. This procedure allows successful isolation, expansion, culture, and mucociliary differentiation of primary bronchial epithelial cells (PBECs) by culturing at the air-liquid interface (ALI) and includes a protocol for biobanking. Furthermore, the characterization of these cultures using cell-specific marker genes is described. These ALI-PBEC cultures can be used for a range of applications, including exposure to whole cigarette smoke or inflammatory mediators, and co-culture/infection with viruses or bacteria. 
The protocol provided in this manuscript, illustrating the procedure in a step-by-step manner, is expected to provide a basis and/or reference for those interested in implementing or adapting such culture systems in their laboratory.

Presented here is a reproducible, affordable, and robust method for the isolation and expansion of primary bronchial epithelial cells for long-term biobanking and the generation of differentiated epithelial cells by culture at the air-liquid interface.

The airway epithelial cell layer forms the first barrier between lung tissue and the outside environment and is thereby constantly exposed to inhaled ...

Collection, Expansion, and Differentiation of Primary Human Nasal Epithelial Cell Models for Quantification of Cilia Beat Frequency

Measurements of cilia function (beat frequency, pattern) have been established as diagnostic tools for respiratory diseases such as primary ciliary dyskinesia. However, the wider application of these techniques is limited by the extreme susceptibility of ciliary function to changes in environmental factors e.g., temperature, humidity, and pH. In the airway of patients with Cystic Fibrosis (CF), mucus accumulation impedes cilia beating. Cilia function has been investigated in primary airway cell models as an indicator of CF Transmembrane conductance Regulator (CFTR) channel activity. However, considerable patient-to-patient variability in cilia beating frequency has been found in response to CFTR-modulating drugs, even for patients with the same CFTR mutations. Furthermore, the impact of dysfunctional CFTR-regulated chloride secretion on ciliary function is poorly understood. There is currently no comprehensive protocol demonstrating sample preparation of in vitro airway models, image acquisition, and analysis of Cilia Beat Frequency (CBF). Standardized culture conditions and image acquisition performed in an environmentally controlled condition would enable consistent, reproducible quantification of CBF between individuals and in response to CFTR-modulating drugs. This protocol describes the quantification of CBF in three different airway epithelial cell model systems: 1) native epithelial sheets, 2) air-liquid interface models imaged on permeable support inserts, and 3) extracellular matrix-embedded three-dimensional organoids. The latter two replicate in vivo lung physiology, with beating cilia and production of mucus. The ciliary function is captured using a high-speed video camera in an environment-controlled chamber. Custom-built scripts are used for the analysis of CBF. Translation of CBF measurements to the clinic is envisioned to be an important clinical tool for predicting response to CFTR-modulating drugs on a per-patient basis.

This protocol describes nasal epithelial cell collection, expansion, and differentiation to organotypic airway epithelial cell models and quantification of cilia beat frequency via live-cell imaging and custom-built scripts.

Measurements of cilia function (beat frequency, pattern) have been established as diagnostic tools for respiratory diseases such as primary ciliary ...

An Air-liquid Interface Bronchial Epithelial Model for Realistic, Repeated Inhalation Exposure to Airborne Particles for Toxicity Testing

For toxicity testing of airborne particles, air-liquid interface (ALI) exposure systems have been developed for in vitro tests in order to mimic realistic exposure conditions. This puts specific demands on the cell culture models. Many cell types are negatively affected by exposure to air (e.g., drying out) and only remain viable for a few days. This limits the exposure conditions that can be used in these models: usually relatively high concentrations are applied as a cloud (i.e., droplets containing particles, which settle down rapidly) within a short period of time. Such experimental conditions do not reflect realistic long-term exposure to low concentrations of particles. To overcome these limitations the use of a human bronchial epithelial cell line, Calu-3 was investigated. These cells can be cultured at ALI conditions for several weeks while retaining a healthy morphology and a stable monolayer with tight junctions. In addition, this bronchial model is suitable for testing the effects of repeated exposures to low, realistic concentrations of airborne particles using an ALI exposure system. This system uses a continuous airflow in contrast to other ALI exposure systems that use a single nebulization producing a cloud. Therefore, the continuous flow system is suitable for repeated and prolonged exposure to airborne particles while continuously monitoring the particle characteristics, exposure concentration, and delivered dose. Taken together, this bronchial model, in combination with the continuous flow exposure system, is able to mimic realistic, repeated inhalation exposure conditions that can be used for toxicity testing.

Described is the cell culture and exposure method of an in vitro bronchial model for realistic, repeated inhalation exposure to particles for toxicity testing.

For toxicity testing of airborne particles, air-liquid interface (ALI) exposure systems have been developed for in vitro tests in order to mimic realistic ...

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

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