Ion transport in epithelial spheroids derived from human airway cells

Abstract

In the present study, we describe a novel three-dimensional airway epithelial explant preparation and demonstrate its use for ion transport studies by electrophysiological technique. Suspension cultures of sheets of epithelial cells released by protease treatment from cystic fibrosis (CF) and non-CF nasal polyps developed free-floating, monolayered epithelial spheres, with the apical, ciliated cell membrane facing the bath and the basolateral cell membrane pointing toward a fluid-filled lumen. Microelectrode impalement of both non-CF and CF spheroids revealed lumen-positive transepithelial electrical potential differences (PDs) that were inhibited by amiloride, indicating that the spheroids were inflated due to amiloride-sensitive Na+absorption followed by water. Transformation to a Cl− secretory state was achieved by addition of ATP to the bath, leading to the development of a diphenylamine-2-carboxylate-sensitive PD. A cAMP-induced increase in PD was seen in non-CF spheroids only. In response to hydrocortisone treatment, Na+ transport reflected by amiloride-sensitive PD increased and more so in CF than in non-CF spheres. We concluded that this preparation is a useful model for the airway surface epithelium and is suitable for studies of transport mechanisms and regulation.

Nasospheroids permit measurements of CFTR-dependent fluid transport

“…The apical membranes face the outside, and basolateral membranes line the lumen of the spherical organoids ( Figure 1). This orientation has been observed by other groups when nondisrupted epithelial sheets are cultured in suspension (40)(41)(42)(43)(44). When CFTR is labeled with a highly specific monoclonal antibody raised against the NBD2 region of CFTR (Ab 528, provided by J.R. Riordan, UNC-Chapel Hill; refs.…”

“…First, we tested non-CF spheroids derived from normal human nasal epithelium (Figure 2). Volume changes in similar spheroids have previously been reported in response to fluid transport changes (40)(41)(42)(43). To test our hypothesis that CFTR activation leads to fluid movement and consequent volume changes of the nasospheroids, we applied forskolin (10 μM) and 3-isobutyl-1-methylxanthine (IBMX,100 μM), a phosphodiesterase inhibitor that maintains intracellular cAMP levels (51), leading to CFTR activation in a cAMP-dependent manner ( Figure 2A).…”

“…Such samples provide a renewable source of differentiated airway epithelial cells from any individual with CF, regardless of their mutation, that does not require tissue expansion and multiple cell divisions prior to performing the assay. To improve the suitability of these samples for both functional and morphological tests, we have developed an organoid culture that is similar to cultures reported previously, where the apical ciliated membrane orients to the media bath (40)(41)(42)(43) but is derived from simple nasal epithelial curettage. This minimally invasive technique does not require polyp excision, which is different from the earlier reports, and utilizes normal nasal epithelial tissue.…”

Polarized Expression of Cystic Fibrosis Transmembrane Conductance Regulator and Associated Epithelial Proteins during the Regeneration of Human Airway Surface Epithelium in Three-Dimensional Culture

“…Several investigators have previously shown that epithelial sheets, dissociated from adult airway surface epithelium, nasal brushings, or nasal polyps and cultured in suspension spontaneously reform free-floating epithelial spheroids in which ciliary activity and ion transport functions could be maintained for several months (Bridges et al, 1991;Jorissen et al, 1991;Pedersen et al, 1999;Ulrich et al, 1998). In all of these studies, the native well-organized surface epithelial cell structures were preserved and no information could therefore be obtained on the depolarization and repolarization process.…”

“…To retain the differentiated and polarized state of the epithelium, several investigators have developed a three-dimensional (3-D) culture model in which epithelial sheets derived from human airway cells form free-floating epithelial spheroids in which ciliary activity and ion transports can be preserved for several months (Jorissen et al, 1989;Pedersen et al, 1999;Ulrich et al, 1998).…”