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Marianne P. Beales, James L. Funderburgh, James V. Jester, John R. Hassell; Proteoglycan Synthesis by Bovine Keratocytes and Corneal Fibroblasts: Maintenance of the Keratocyte Phenotype in Culture. Invest. Ophthalmol. Vis. Sci. 1999;40(8):1658-1663.
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purpose. To determine the effect of serum on morphology, growth, and
proteoglycan synthesis by primary cultures of collagenase-isolated
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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