Physiological 3D tissue model of the airway wall and mucosa

Abstract

This protocol describes the setup, maintenance and characteristics of a tissue-engineered model of the human bronchial mucosa that can be used for basic physiology and pathophysiology studies. The model includes a well-differentiated epithelium with functional cilia, mucus secretion and subepithelial fibroblasts within type I collagen. The tissue is created within porous polymeric wells to prevent gel contraction and allow culture at the air-liquid interface. It requires at least 2 wk to be established and can be maintained thereafter for over 4 wk, with tissue differentiation moving towards a more physiologically relevant phenotype with increasing time in culture. Over time, the extracellular matrix also remodels, depositing proteins such as types III and IV collagen and fibronectin. Because it recapitulates many key anatomical and functional features of the airway wall, this model is well suited for a wide range of studies, including those on airway remodeling, transepithelial transport and inflammatory cell interactions with the mucosa. The entire protocol takes 4-6 wk, including cell expansion, depending on the extent of ciliogenesis desired.

Non-animal models of epithelial barriers (skin, intestine and lung) in research, industrial applications and regulatory toxicology

“…Additionally, the device is built of flexible materials, which allow controlled stretching of the cells on the membrane to simulate the cyclical stretch of the lung tissue during inhalation and exhalation. Other models have used tissue engineered matrices to support airway cells in a three-dimensional tissue equivalent (Choe et al, 2006). Using biocompatible hydrogels as an ECM analogue offers the advantage of modulating the ECM stiffness, which is of interest in asthma remodeling processes.…”

<em>Ex vivo</em> Method for High Resolution Imaging of Cilia Motility in Rodent Airway Epithelia

“…Such coordinated ciliary motion is not observed in reciliating mouse tracheal epithelial cultures, which often ciliate poorly and show cilia beat in random directions 9,10 . The coordinated ciliary beat observed using the tracheal epithelia technique described here can provide the means to interrogate the mechanisms regulating ciliary beat coordination and its potential role in mucociliary clearance.…”

“…Acute experiments include ex vivo visualization of human nasal/airway brush biopsies 6,7 and analysis of simple transverse airway sections 8 . The in vitro approaches utilize various cell culture techniques to generate sheets of differentiated ciliated epithelia such as in air liquid interface cultures or airway suspension cultures [9][10][11] . However, these airway epithelia reciliation techniques require very significant investment in time and training before any useable ciliated epithelial cells are produced for experimentation (4-6 weeks 9,10 ).…”

“…The in vitro approaches utilize various cell culture techniques to generate sheets of differentiated ciliated epithelia such as in air liquid interface cultures or airway suspension cultures [9][10][11] . However, these airway epithelia reciliation techniques require very significant investment in time and training before any useable ciliated epithelial cells are produced for experimentation (4-6 weeks 9,10 ). While acute ex vivo analysis of airway epithelial brush biopsies are commonly used for human clinical studies, this method is not usable in mouse studies due to exacerbated mechanical tissue injury 12 .…”

Dendritic cell functional properties in a three-dimensional tissue model of human lung mucosa

“…More recently, protocols describing the setup of bronchial 3D mucosal tissue models that better recapitulate human lung mucosa have been developed (6). This includes models with a physiologically relevant fibroblast matrix layer and epithelial differentiation into ciliated, mucus-secreting cells (6,9).…”