DEFINED MEDIUM FOR PRIMARY CULTURE DE-NOVO OF ADULT-RAT ALVEOLAR EPITHELIAL-CELLS

Isolated type II pneumocytes grown in serum on tissue culture-treated polycarbonate filters form monolayers with characteristic bioelectric properties, and change morphologically with time in culture to resembl e type I cells. Concurrently, the cells express type I cell surface ep itopes, making this a potentially useful in vitro model with which to study regulation of alveolar epithelial cell function and differentiat ion. To define specific soluble growth factors and matrix substances t hat may regulate these processes, it would be preferable to culture is olated pneumocytes de novo under completely defined, serum-free condit ions. In this study, we developed a completely defined serum-free medi um that is capable of supporting alveolar epithelial cells in primary culture, allowing the formation of monolayers with characteristic bioe lectric and phenotypic properties. Freshly isolated rat type II cells were resuspended in completely defined serum-free medium and plated de novo on polycarbonate filters. Plating efficiency, bioelectric proper ties, morphology, and binding of a type I cell-specific monoclonal ant ibody were determined as functions of time. Plating efficiency plateau s at about 14% by Day 3 in culture. Transepithelial resistance rises t o high levels, peaking at 1.76 +/- 0.14 K Omega-cm(2) by Day 5 in cult ure. Short-circuit current peaks on Day 3 in culture at 2.71 +/- 0.35 mu A/cm(2). With time, the cells gradually become flattened with protu berant nuclei and long cytoplasmic extensions, more closely resembling type I cells, and begin to express a type I cell surface epitope. The se observations indicate that it is feasible to culture alveolar epith elial cell monolayers under completely defined serum-free conditions d e novo, This culture system should prove useful for identifying solubl e growth factors and matrix substances that modulate alveolar epitheli al cell biological properties.