Characterization of human pulmonary endocrine cells maintained in vitro

Endocrine cells located in the epithelium of human fetal airways contain the amine, 5-hydroxytryptamine (5HT), and the peptide, bombesin (BOM), but difficulties in studying these cells experimentally have slowed progress in understanding their functional roles. This investigation describes an in vitro method to maintain pulmonary neuroendocrine cells (PNEC) in organ culture. Bronchial trees from human fetal lungs were dissected free of adherent blood vessels and lung tissue. Explants of the airways were placed in culture dishes containing defined tissue culture medium for five days. Using indirect immunofluorescence, 5HT- and BOM-like immunoreactive cells were observed both in nonincubated airways and in explants maintained for five days in organ culture. The number of 5HT-immunoreactive cells/0.1 mm2 of airway epithelium was not significantly different in the two groups, although there was a significant reduction in 5HT content measured by HPLC after the five-day culture period. The diameter of dense core vesicles and the number of dense core vesicles/micron2 of endocrine cell cytoplasm in cultures were not significantly different from non-incubated controls. Treatment of the explants with the 5HT-synthesis inhibitor p-chlorophenylalanine resulted in a significant reduction both in the number of 5HT-containing cells/0.1 mm2 of airway epithelium and in the 5HT content. These results demonstrate that both 5HT and BOM content in endocrine cells of explants from human fetal airways can be well maintained in organ culture for at least 5 days and that they are responsive to pharmacologic inhibition of 5HT synthesis.