1774 FETAL LUNG EPITHELIAL CELLS CULTURED ON FIBROBLAST FEEDER LAYER

Abstract

Mouse embryo fibroblasts (line 3T3) elaborate an extracellular matrix comprising macromolecules essential for attachment and growth of various types of differentiated cells. We investigated whether a feeder layer of these fibroblasts would promote growth of lung epithelial cells. Epithelial cells were isolated from lungs of mature fetal rabbits (28-29 d gestation) by either ex-plant culture or exposure to 0.25% trypsin followed by differential adherence. The cells were cultured in Dulbecco's Modified Eagle's medium + 10% fetal bovine serum on either plastic or a feeder layer of 3T3 fibroblasts rendered mitotically inactive by treatment with mitomycin C. Epithelial cells cultured on plastic degenerated and died within 7 d. In contrast, cells cultured on a feeder layer could be passaged weekly for 10 wk and underwent 6 population doublings. Light microscopy showed that spindle-shaped 3T3 fibroblasts surrounded colonies of cuboidal lung cells. The latter were Judged to be type 2 epithelial cells on the basis of the following ultrastructural criteria: cuboidal shape, presence of microvilli, tonafilaments, and characteristic lamellar bodies (during first wk). The cells contained organelles indicative of a high level of biosynthesis (rough endoplasmic reticulum, Golgi apparatus) and numerous mitochondria, and they formed junctions. Multivesicular lamellar bodies were present in late cultures (4–8 wk). These results demonstrate that a feeder layer of 3T3 fibroblasts can support extended culture of fetal lung epithelial cells.