Fig. 2

Isolation and growth of the hSABCi-NS1.1 immortalized small airway basal cells from a healthy nonsmoker subject. a Morphology of cells following drug selection, day 9. Left - parental cells without TERT, plus puromycin selection; right - hTERT infected cells with puromycin selection (mixed clones, hSABCi-NS1). Bar = 100 μm. b Morphology of the single cell-derived cell clone hSABCi-NS1.1 on day 6, passage 0. Single-cell clones were generated by culturing limited-numbers of cells of hSABCi-NS1 cell in 10 cm plate and tracked under the microscope from day 2. Bar = 100 μm. c Growth of hSABCi-NS1.1. Cell population doubling levels were quantified from passage 5. Each dot represents one passage in T25 flask. d Growth of hSABCi-NS1.1 after recovery from cryopreservation. Passage 46 of hSABCi-NS1.1 cells (red dots) which had been frozen in the liquid nitrogen for 131 days, was re-cultured to test the consistence of growth. The population doubling levels were re-counted from the day of re-culturing. Passage 1 parental pre-immortalized primary cells were cultured at the same time as control (blue triangles). e % live cells of the re-cultured cells. The % live cells of the re-cultured hSABCi-NS1.1 cells were counted during each passage. Only adherent cells were counted. Red – hSABCi-NS1.1, passage 46; blue – parental primary cells pre-TERT, passage 2. f Growth of the re-cultured hSABCi-NS1.1 cells at passage 52. Cell doubling time between day 2 and day 3 after seeding was 16 h. The rate of cell doubling was reduced after day 5 when the cells reached confluence