Ljv. Galietta et al., AN IMPROVED METHOD TO OBTAIN HIGHLY DIFFERENTIATED MONOLAYERS OF HUMAN BRONCHIAL EPITHELIAL-CELLS, In vitro cellular & developmental biology. Animal, 34(6), 1998, pp. 478-481
Electrophysiological studies of human bronchial epithelial cells in vi
tro are limited by the scarcity of biological material available for p
rimary culture. To overcome this problem, vie set up a protocol in whi
ch the cell number is first enlarged in LHC9/RPMI 1640 serum-free medi
um for up to six passages, each passage giving a four- to eightfold am
plification. The cells are then plated at high density on permeable su
pports. Cell differentiation, monitored by measuring transepithelial p
otential difference (PD) and electrical resistance (R), is induced wit
h a medium containing serum and a cocktail of different supplements an
d hormones. Maximal values of PD and R, obtained after 4-7 d of cultur
e on permeable supports, are around - 50 mV and 3000-4000 Ohm/cm(2), r
espectively. Ussing chamber experiments show that basal short-circuit
current (I-sc) is partially inhibited by the epithelial Na+ channel bl
ocker amiloride. Stimulation with a cAMP-elevating agent induces a I-s
c increase that is inhibited by the cystic fibrosis transmembrane cond
uctance regulator (CFTR) blocker glibenclamide. Our culture protocol p
rovides a large number of differentiated bronchial epithelial cell mon
olayers starling from a low amount of material. This characteristic is
useful for in vitro studies of ion transport in airway epithelium.