Sphere-Forming Culture for Propagation

For sphere-forming cultures, cells were plated at a density of 2 × 10⁴ cells per well in six-well ultra-low attachment plates (Corning Inc., Corning, NY) in serum free Bronchial Epithelial Cell Basal Medium (BEBM), supplemented with the following growth supplements: BPE, Hydrocortisone, hEGF, Epinephrine, Transferrin, Insulin, Retinoic Acid, Triiodothyronine, and GA-1000, provided as BEGM prepackaged SingleQuots (Lonza, Walkersville, MD) plus rhEGF (Sigma, St Louis, MO), bFGF (Sigma). Fresh aliquots of EGF and bFGF were added every three days. Floating spheres were subjected to mechanical dissociation, followed by replating of single cells for propagation into a second, and subsequently into a third batch of suspension culture as described in our previous reports [5 (link)-7 (link)].