Figure 2.

Schematic overview of nasal cell expansion in EpiX^TM medium and Ussing chamber protocol. (A) Nasal epithelial cells were collected from five CF individuals by nasal brushings. Isolated primary cells were expanded with the EpiX^TM method in Keratinocyte-SFM medium with TGF-β inhibitor A83-01 (1 uM), Rho-Kinase inhibitor Y-27632 (5 uM), isoproterenol (3 uM), and low calcium concentrations (CaCl₂; 90 uM), as previously described (6). Expanded cells were plated on Snapwell inserts at Passage 3 or 4 and subsequently grown in a serum-free medium at an air-liquid interface for 21–28 days for differentiation. (B) Population doublings. The initial “lag phase” illustrates the number of days it took to generate CF airway cultures (passage 1) from the primary cells (P0) that were isolated from the brushed tissue sample. Each CF culture reached ~25 population doublings within 25–35 days. (C) Activities for ENaC, CFTR, and CaCC were assessed in non-treated cultures and cultures treated with CFTR corrector VX-809 or VX-661. CFTR activity was assessed by measuring cAMP-dependent activation by forskolin followed by acute addition of CFTR potentiator VX-770. CFTR inhibitor compounds were used to identify CFTR-mediated chloride currents. Epithelial Na channel activity was quantified by amiloride. Calcium-activated chloride currents (CaCC) were activated by ATP and measured in the presence of CFTR inhibitors.