Fluid absorption related to ion transport in human airway epithelial spheroids

Abstract

Airway epithelium explants from cystic fibrosis (CF) patients and non-CF subjects formed monolayered spheres, with the apical ciliated cell membrane facing the bath and the basolateral cell membrane pointing toward a fluid-filled lumen. With the use of two microelectrodes, transepithelial potential difference and changes in potential difference in response to passage of current pulses were recorded, and epithelial resistance and the equivalent short-circuit current were calculated. Non-CF control potential difference and short-circuit current values were significantly lower than the CF values, and amiloride inhibited both values. Fluid transport rates were calculated from repeated measurements of spheroid diameters. The results showed that 1) non-CF and CF spheroids absorbed fluid at identical rates (4.4 microl x cm(-2) x h(-1)), 2) amiloride inhibited fluid absorption to a lower residual level in non-CF than in CF spheroids, 3) Cl(-)-channel inhibitors increased fluid absorption in amiloride-treated non-CF spheroids to a level equal to that of amiloride-treated CF spheroids, 4) hydrochlorothiazide reduced the amiloride-insensitive fluid absorption in both non-CF and CF spheroids, and 5) osmotic water permeabilities were equal in non-CF and CF spheroids ( approximately 27 x 10(-7) cm x s(-1) x atm(-1)).

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.…”

Water permeability in human airway epithelium

“…A human airway epithelial 3D preparation, with morphological and functional features resembling those of natural airway epithelia, was recently described [6,30,31]. Suspension of protease-released small fragments of surface epithelium from resected nasal polyps (from normal subjects and from CF patients) formed monolayered, sphere-shaped and fluid-filled explants of ciliated airway epithelium [30,31] (Fig.…”

“…Suspension of protease-released small fragments of surface epithelium from resected nasal polyps (from normal subjects and from CF patients) formed monolayered, sphere-shaped and fluid-filled explants of ciliated airway epithelium [30,31] (Fig. 1).…”