Improved maintenance of adult rat alveolar type II cell differentiation in vitro: effect of serum-free, hormonally defined medium and a reconstituted basement membrane.

We have developed a serum-free, hormonally defined medium for maintenance of differentiation of adult type II cells cultured on Engelbreth-Holm-Swarm (EHS) tumor basement membrane gels. This defined medium consists of 1:1 (vol/vol) mixture of Ham's F12 and Dulbecco's modified Eagle's media supplemented with insulin, dibutyryl cyclic AMP, hydrocortisone, epidermal growth factor, selenium, and albumin/linoleic acid complex. Compared to cells cultured on EHS gels in serum-supplemented medium, type II cells cultured on EHS gels in this defined medium showed increased acetate incorporation into total lipids (10-fold) and an increase in the relative percentage of acetate incorporated into phosphatidylcholine (PC) (87.8 +/- 0.4% versus 78.5 +/- 1.0% [mean +/- SE]; P less than 0.01), saturated phosphatidylcholine (SPC) (61.4 +/- 0.5% versus 55.2 +/- 0.9%; P less than 0.01), and phosphatidylglycerol (PG) (5.3 +/- 0.3% versus 0.8 +/- 0.1%; P less than 0.01) and decreased acetate incorporation into neutral lipids (9.7 +/- 0.8% versus 62.6 +/- 1.9%; P less than 0.01). No response to this defined medium was seen when type II cells were cultured on tissue culture plastic. Type II cells cultured on EHS gels in serum-supplemented medium for 4 d had numerous neutral lipid droplets in their cytoplasm. In contrast, neutral lipid droplets were not commonly observed within the cytoplasm of the cells cultured in serum-free, hormonally defined medium on EHS gels. This morphologic finding was consistent with the result that cells cultured in serum-supplemented medium significantly increased the relative percentage of acetate incorporated into neutral lipids. These data indicate that adult type II cells cultured on a reconstituted basement membrane (EHS gels) can be maintained in synthetic culture medium without serum. These culture conditions permit the expression of a pattern of differentiated phospholipid biosynthesis and cell morphology more similar to normal type II cell differentiation.