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(Investigative Ophthalmology and Visual Science. 1999;40:1658-1663.)
© 1999 by The Association for Research in Vision and Ophthalmology, Inc.

Proteoglycan Synthesis by Bovine Keratocytes and Corneal Fibroblasts: Maintenance of the Keratocyte Phenotype in Culture

Marianne P. Beales1,2, James L. Funderburgh3, James V. Jester4 and John R. Hassell1,2

1 From the Research Department, Shriners Hospitals for Children, Tampa, Florida; the 2 Department of Biochemistry and Molecular Biology, College of Medicine, University of South Florida, Tampa; the 3 Division of Biology, Ackert Hall, Kansas State University, Manhattan; and the 4 University of Texas, Southwestern Medical Center, Dallas.

PURPOSE. To determine the effect of serum on morphology, growth, and proteoglycan synthesis by primary cultures of collagenase-isolated bovine keratocytes.

METHODS. Keratocytes were isolated from bovine corneas using sequential collagenase 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 metabolically radiolabeled with 35SO4. The proteoglycans were characterized by their sensitivity to keratanase, chondroitinase ABC, and heparatinase and 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 fibroblastic, and synthesized only 9% of the total glycosaminoglycan as keratan sulfate (KS), whereas cells in serum-free media were quiescent, appeared dendritic, and synthesized 47% KS, a value similar to the 45% KS for corneas radiolabeled overnight in organ culture. This increased proportion of KS synthesis in serum-free media was caused by a moderate increase in KS synthesis combined with a substantial decrease in chondroitin sulfate (CS) synthesis. Fractionation on Superose 6 High Resolution showed the size and relative amounts of the CS- and KS-containing proteoglycans synthesized by keratocytes in serum-free media also more closely resembled that of keratocytes in corneas in organ culture than keratocytes in media containing serum.

CONCLUSIONS. A comparison of proteoglycan synthesis and cell morphology between keratocytes in corneas in organ culture and in cell culture indicates that keratocytes maintain a more native biosynthetic phenotype and appearance when cultured in serum-free media. These results also suggest that culturing in the presence of serum fundamentally alters the keratocyte phenotype to an activated cell, mimicking certain changes observed during wound healing.




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