1735 A SERUM-FREE SYSTEM FOR THE STUDY OF MITOGEN EFFECTS ON TYPE PNEUM0CYTES IN PRIMARY CULTURE

Abstract

Isolated type II pneumocytes are reported not to divide in primary culture, and the cell number usually falls with time in culture. A serum-free culture system has been developed which allows the response of a stable type II pneumocyte population to exogenous mitogens to be studied over several days in culture.Type II pneumocytes ware isolated from adult rat lung. After a 36 hr plating period cell number, purity (>90%) and viability (>95%) remained stable over 7 days in a serum-free medium supplemented with fibronectin, insulin, transferrin and selenous acid (F.I.S.T.). Plating efficiency was 15-25%. Under serum-free conditions pneumocytes increase in number (p<.01) in response to EGF 50ng/ml, though there is a subsequent loss of adherent cells. The response to EGF is lost in the presence of 10% fetal bovine serum. The addition of 10% adult rat plasma, however, allows a sustained increase in cell number over 9 days in culture (p<.05).After a brief period of conditioning with rat serum, type II pneumocytes maintained in serum-free medium respond to F.I.S.T. by a 60% increase in cell number over 8 days in culture. These studies have shown that type II pneumocytes can be maintained under serum-free conditions for several days in primary culture, and respond to known mitogens by an increase in cell number.