hAE cells from the second or third passage were seeded onto a
Millicell cell culture plate insert (Millipore Corp., Bedford, MA,
USA) in a 24-well culture plate at a concentration of
1.0 105 cells/cm2. The hAE cells were cultured while submerged
for 4 days then grown at an air–liquid interface for 3
weeks with serum-free MCDB 153 medium containing a high
calcium concentration of 1.8 mmol/L. The medium was
changed every 2 days.