Maintenance of differentiated phenotype by mouse type 2 pneumocytes in serum-free primary culture

An improved method has been developed for separation of an enriched population of mouse type 2 pneumocytes, based on differential adherence and size fractionation of cells dissociated with trypsin. These cells were successfully maintained in primary culture in serum-free medium MCDB 201 supplemented with albumin, transferrin, and lipids. Whereas type 2 pneumocytes in serum-supplemented culture undergo phenotypic transformation into adherent flattened cells that resemble type 1 pneumocytes, this did not occur in serum-free culture. Both the morphology of the type 2 pneumocytes and their expression of surfactant protein A were maintained for at least 6 days in vitro. However, rapid loss of differentiated characteristics was induced by exposure of the cells to normal mouse serum. This was accompanied by a striking decrease in spontaneous DNA synthesis as assessed by incorporation of tritiated thymidine. When cultured in serum-free medium, the behavior of the type 2 pneumocytes on various extracellular matrix components was different from that reported for serum-supplemented culture. Serum-free culture of type 2 pneumocytes may offer significant advantages for evaluation of the secretory activities of these cells in vitro.