Development of a bioassay for pulmonary cell production of fibrogenic factors

Summary: Fibroblast proliferation and enhancement of collagen synthesis are key steps in the development and progression of pulmonary fibrosis. The current investigation presents a fibroblast proliferation assay to detect the release of competence factors from pneumocytes. The specific objective of this study was to define cell culture conditions for fibroblasts that would allow selective detection of competence factors in test media. The results indicate the following: (a) fibroblasts should be cultured for 2 days in plasma-free medium rather than 2% plasma to assure complete quiescence; (b) 1% plasma was optimum for producing progression activity for the bioassay of a competence factor, while 0.5% plasma did not produce sufficient progression activity (yielding false negatives) and 2% plasma often contained significant competence activity (yielding false positives); and (c) fresh plasma should be used since the progression activity of plasma was adversely affected by freezing and thawing. Under these conditions, this bioassay can be employed with either rat or human lung fibroblasts to detect competence factors, such as platelet-derived growth factor (PDGF), at levels as low as 25 ng/ml.