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 po pulation 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 M CDB 201 supplemented with albumin, transferrin, and lipids. Whereas ty pe 2 pneumocytes in serum-supplemented culture undergo phenotypic tran sformation into adherent flattened cells that resemble type I pneumocy tes, this did not occur in serum-free culture. Both the morphology of the type 2 pneumocytes and their expression of surfactant protein A we re maintained for at least 6 days in vitro. However, rapid loss of dif ferentiated characteristics was induced by exposure of the cells to no rmal mouse serum. This was accompanied by a striking decrease in spont aneous DNA synthesis as assessed by incorporation of tritiated thymidi ne. When cultured in serum-free medium, the behavior of the type 2 pne umocytes on various extracellular matrix components was different from that reported for serum-supplemented culture. Serum-free culture of t ype 2 pneumocytes may offer significant advantages for evaluation of t he secretory activities of these cells in vitro.