Maintenance of differentiated function of the surfactant system in human fetal lung type II epithelial cells cultured on plastic

We report a simplified culture system for human fetal lung type II cells th at maintains surfactant expression. Type II cells isolated from explant cul tures of hormone-treated lungs (18-22 wk gestation) by collagenase + trypsi n digestion were cultured on plastic for 4 days in serum-free medium contai ning dexamethasone (Dex, 10 nM) + 8-bromo-cAMP (0.1 mM) + isobutylmethylxan thine (0.1 mM) or were untreated (control). Surfactant protein (SP) mRNAs d ecreased markedly in control cells between days 1 and 4 of culture, but mRN A levels were high in treated cells on day 4 (SP-A, SP-B, SP-C, SP-D; 600%, 100%, 85%, 130% of day 0 content, respectively). Dex or cAMP alone increas ed SP-B, SP-C, and SP-D mRNAs and together had additive effects. The greate st increase in SP-A mRNA occurred with cAMP alone. Treated cells processed pro-SP-B and pro-SP-C proteins to mature forms and had a higher rate of pho sphatidylcholine (PC) synthesis (2-fold) and higher saturation of PC (simil ar to 34% versus 27%) than controls. Only treated cells maintained secretag ogue-responsive phospholipid synthesis. By electron microscopy, the treated cells retained lamellar bodies and extensive microvilli. We conclude that Dex and cAMP additively stimulate expression of surfactant components in is olated fetal type II cells, providing a simplified culture system for inves tigation of surfactant-related, and perhaps other, type II cell functions.