Proteoglycan synthesis by bovine keratocytes and corneal fibroblasts: Maintenance of the keratocyte phenotype in culture

PURPOSE. To determine the effect of serum on morphology, growth, and proteo glycan synthesis by primary cultures of collagenase-isolated bovine keratoc ytes. METHODS. Keratocytes were isolated from bovine corneas using sequential col lagenase digestion and cultured in Dulbecco's modified Eagle's medium (DMEM ), with and without fetal bovine serum (FBS). Proteoglycans synthesized by the cells in culture and by keratocytes in intact cornea culture were metab olically radiolabeled with (SO4)-S-35. The proteoglycans were characterized by their sensitivity to keratanase. chondroitinase ABC, and heparatinase a nd by their size on Superose 6 HR. Cell number was determined by measuring DNA content of the culture dishes. RESULTS. Keratocytes cultured in 10% FBS proliferated, appeared fibroblasti c, and synthesized only 9% of the total glycosaminoglycan as keratan sulfat e (KS), whereas cells in serum-free media were quiescent, appeared dendriti c, and synthesized 47% RS, a value similar to the 45% KS for corneas radiol abeled overnight in organ culture. This increased proportion of KS synthesi s in serum-free media was caused by a moderate increase in KS synthesis com bined with a substantial decrease in chondroitin sulfate (CS) synthesis. Fr actionation on Superose 6 High Resolution showed the size and relative amou nts of the CS- and KS-containing proteoglycans synthesized by keratocytes i n serum-free media also more closely resembled that of keratocytes in corne as in organ culture than keratocytes in media containing serum. CONCLUSIONS. A comparison of proteoglycan synthesis and cell morphology bet ween keratocytes in corneas in organ culture and in cell culture indicates that keratocytes maintain a more native biosynthetic phenotype and appearan ce when cultured in serum-free media. These results also suggest that cultu ring in the presence of serum fundamentally alters the keratocyte phenotype to an activated cell, mimicking certain changes observed during wound heal ing.