Growth of distal fetal rat lung epithelial cells in a defined serum-free medium

Fetal rat distal lung epithelial cells, in contrast to adult type II pneumocytes, will divide readily in culture in the presence of 10% (vol:vol) fetal bovine serum. The presence of serum makes purification of uncontaminated cell-derived growth factors difficult and modifies cellular responses to oxidant injury. We report the development of a defined serum-free medium that will support growth of fetal distal lung epithelial cells in primary culture. Initial studies used a low-serum (2%; vol:vol) to determine the effect of basal media, substrata, and various additives. Subsequent studies demonstrated growth on a poly-D-lysine substratum under serum-free culture conditions in Dulbecco's modified minimal essential medium with insulin (50 micrograms/ml), endothelial cell growth supplement (20 micrograms/ml), bovine pituitary extract (100 micrograms/ml), bovine serum albumin (50 micrograms/ml), selenous acid (4 ng/ml), reduced glutathione (500 ng/ml), soybean trypsin inhibitor (100 micrograms/ml), transferrin (5 micrograms/ml), HEPES buffer (2.6 mg/ml), and cholera toxin (5 micrograms/ml). Growth was enhanced by reducing the gas phase oxygen concentration from 21 to 3%. The undefined components of this medium, bovine pituitary extract and endothelial cell growth supplement, could be replaced by platelet-derived growth factor (20 ng/ml) with prostaglandin E1 (25 nM). The response of fetal distal lung epithelial cells to known growth factors differs substantially from that observed with type II pneumocytes from adult lung and is similar in many, though not all, respects to the responses reported for proximal airway cells from adult lung.