Determining Ciliary Function and Membrane Impermeability of the Pseudostratified Lung Airway Epithelium

Summary

Air-liquid interface culture is commonly used to develop pseudostratified airway epithelium by differentiating primary normal human bronchial epithelial cells that mimic the apical side of the lung airway. Here, we describe an easy protocol for determining its quality by monitoring its biophysical properties, such as ciliary function and membrane integrity.

Abstract

Differentiating primary lung airway epithelial cells in the air-liquid interface (ALI) is a popular technique to develop a multi-cellular pseudostratified airway epithelium that mimics the apical side of the lung airway. While the differentiation of primary lung airway cells is expected, the assessment of biophysical properties like ciliary function and membrane impermeability provides a quality assessment of the airway epithelium and ensures the reliability of the experiment. Here, we describe a straightforward protocol for the development of multi-cellular pseudostratified airway epithelium in ALI culture and assess two important biophysical properties: ciliary function and membrane impermeability. To determine ciliary function, we captured the ciliary movement of a 4-week differentiated airway epithelium using a high-speed camera attached to an inverted microscope, followed by quantifying ciliary beat frequency (CBF) using the Simon Amon Video Analysis (SAVA) system. We also measured transepithelial electrical resistance (TEER) using a volt-ohm meter to determine the epithelial barrier integrity of the airway epithelium.

Introduction

Chronic respiratory diseases such as asthma, chronic obstructive pulmonary disease (COPD), and cystic fibrosis are some of the major health concerns worldwide¹. The prevalence of respiratory diseases caused by viruses such as human influenza A virus, respiratory syncytial virus (RSV), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) also cause economic and public health burden². Therefore, there is an immense need to develop treatment regimens for respiratory illnesses that lead to irreversible respiratory tissue damage. The respiratory epithelial tissue itself is not only involved in oxygen uptake but al....

Protocol

The protocol should be performed in a sterile condition under a biosafety level 2 laminar flow hood (biosafety cabinet). All the medium and solutions should be thawed at 37 °C prior to use. Centrifugation should be performed at 4 °C. All the materials used in the experiment must be sterile. The primary cells (deidentified donor) were obtained in collaboration with Dr. Kristina Bailey at the University of Nebraska Medical Center (UNMC), Omaha, NE. The primary cells are from deidentified donors, and the cells do not fall under the NIH Human Subject. The cells were obtained under an approved Material Transfer Agreement (MTA) between the University of North Dako....

Results

We evaluated both ciliary function and membrane impermeability of the airway epithelium obtained from differentiating primary NHBE cells from two independent deidentified donors (two nonsmoking, healthy adults or adults with COPD, above 50-year-old females). For determining ciliary function, we determined the CBF of the 28-day differentiated airway epithelium. We observed CBF reached at least 3 Hz for both airway epithelium, which was comparable to the previously published results (Figure 1).......

Discussion

Human primary airway epithelium (ALI culture) is increasingly used as an in vitro lung model for biological assessments to investigate function and mechanisms and to reduce animal use¹⁴. ALI has several advantages over any monolayer submerged culture. For example, it provides a tissue-like pseudostratified airway epithelium that mimics the apical side of the lung airway^6,7,14. However, the quality and int.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1x DPBS	Thermo Fisher Scientific, USA	14190-144
Airway Epithelial Cell Growth Medium	PromoCell, Germany	C-21060
Amphotericin B	Thermo Fisher Scientific, USA	15290-026
EVOM2 volt Ohmmeter	World Precision Instruments, USA	Not applicable
Heparin Solution	STEMCELL technologies, USA	07980
Hydrocortisone stock	STEMCELL technologies, USA	07926
Microscope: Leica DMI8 (Leica Microscope) with 120f/sec high-speed camera (Basler) associated with the microscope. Microsope also fixed with stage chamber incubator i8.	Leica, USA	Not applicable
Penicillin-Streptomycin	Corning, USA	30-002-CI
PneumaCult-ALI Basal Medium	STEMCELL technologies, USA	05002
PureCol	Advanced BioMatrix, USA	5005
Sisson-Ammons video analysis (SAVA) system. Software V.2.1.15	Amson Engineering, USA	Not applicable