PROLIFERATION OF RAT ALVEOLAR EPITHELIAL-CELLS IN LOW-DENSITY PRIMARYCULTURE

Alveolar type II cells proliferate to restore the alveolar epithelium after lung injury and differentiate into type I epithelial cells. A va riety of factors promote rat type II cell DNA synthesis in vitro; howe ver, only low levels of proliferation occur when type II cells are cul tured at high density. We plated type II cells at low density to deter mine if those growth factors that stimulate thymidine incorporation al so stimulate low density proliferation. Type II cells were plated at 1 x 10(3) cells/cm2 in Dulbecco's modified Eagles medium containing 2 % fetal bovine serum, cholera toxin, insulin, epidermal growth factor, acidic fibroblast growth factor (aFGF), and concentrated bronchoalveol ar lavage-fluid from normal rats. By 7 days, numerous colonies had gro wn out that exhibited an epithelial morphology and stained positively for cytokeratin. The cell number at day 7 in the presence of the combi ned factors was 5.9 x 10(3) (+/- 0.6 x 10(3)) cells/cm2 (n = 4). There was no colony formation in the absence of fetal bovine serum. The add ition of linoleic acid to serum-free medium containing all the growth supplements was found to partially restore colony formation. When aFGF or lavage fluid was omitted from the culture medium, colony formation was dramatically reduced. The colonies lacked characteristics of diff erentiated type II cells, which was anticipated since these cells were cultured on tissue culture plastic. To see if these cells could expre ss differentiated functions, we maintained the colonies under growth c onditions, removed them from the plastic substratum, and then replated them on EHS matrix. Under these conditions, the epithelial cells expr essed mRNAs for surfactant protein (SP)-A, SP-B, and SP-C, contained S P-A and SP-D protein by immunocytochemistry, and exhibited surface alk aline phosphatase by enzyme histochemistry. These results demonstrate that proliferation of adult rat alveolar epithelial cells can be achie ved in vitro and that aFGF and a factor(s) from bronchoalveolar lavage fluid are important in this response.