Figure 5
Expansion and differentiation of non-CF and CF nasal epithelial cells. (A) Ciliated cells (black arrows) are observed in freshly isolated CF nasal cultures by phase contrast microscopy. Bar, 20 μm. (B) Timeline for processing of nasal curettage samples showing frozen cell banking (F), plating for differentiation on Transwell permeable supports (T) or plating for expansion in FYRM submerged culture. Due to the small initial sample size, cells were not banked or plated for differentiation until P4. Cells beyond P7 are not typically used to generate differentiated cultures for experimental analysis. Detail related to culture on Transwells (T) is shown in Fig. 2. (C) Non-CF and CF nasal epithelial cells with 3 different genotypes had a comparable doubling time when cultured in CRC conditions. n = 3 – 6 wells for CF cells; doubling data for NhNE cells is from Fig. 2. (D, E) Cilia and mucus producing CF nasal epithelial cells as observed by scanning electron microscopy at 500x (D) and 2000x (E) magnification. (F–H) Immunofluorescence confocal microscopy of CF nasal airway cells showed tight junctions (green, ZO-1) and cilia (red, Ac-Tubulin). Nuclei were labeled with DAPI (blue). Bar, 20 μm (F, H).
