Keratocyte Growth in Media Containing Defined Growth Factors

Abstract

Abstract: : Purpose:We showed that keratocytes cultured in defined media containing certain purified growth factors are stimulated to incorporate ³H–thymidine into DNA. Here we compare the effects of purified growth factors on prolonged cell culture and maintenance of the keratocyte marker proteins keratocan and ALDH during proliferation. Methods: Keratocytes were isolated from bovine corneas and plated in DMEM at 5,000 cells/cm2. The medium was changed on day 1 to DMEM alone or DMEM supplemented with either: PDGF–BB (50ng/ml); FGF–2 (10ng/ml); IGF–1 (10 ng/ml), insulin (10 ug/ml) or ITS. Cells were radiolabeled with ³H–thymidine (20 uCi/ml DMEM) or ³⁵SO₄ (50 uCi/ml DMEM) from day 1 to day 4 and from day 4 to day 7 and incorporation measured by liquid scintillation. DNA content was measured using Cyquant and incorporation determined per ng DNA. ALDH in the cells and keratocan in the media were measured by western blotting using antisera to keratocan’s core protein and antisera to ALDH (gift of Dr. R. Lindahl). ALDH content and keratocan accumulation were also measured per ng DNA. The size of the proteoglycans was evaluated by chromatography on Superose 6. Cell morphology was analyzed by digital photography. Results:Culture in all growth factors resulted in growth or at least maintenance of original cell density and increased ³H–thymidine incorporation. Analysis for ALDH shows that all growth factors maintained ALDH content over the 7–day culture period. Compared to DMEM on day 4, the insulin containing medium ITS as well as PDGF–BB increase keratocan levels in the media 28.47 fold and 15.67 fold, respectively. On day 7 there was a marked increase of keratocan in the media of cells cultured in insulin (118.69–fold), ITS (219.46 fold) and PDGF–BB (410.88 fold). Sulfate incorporation, however, was not stimulated by any of the growth factors on day 4 or day 7. Most of the incorporated ³⁵SO₄ from the DMEM cultures eluted on Superose 6 after the PDGS marker in the low MW range (free KS chains) while most of the incorporated ³⁵SO₄ in the ITS, insulin and the PDGF–BB cultures eluted with the keratocan marker in the high MW range. Keratocytes cultured in IGF–1, insulin and ITS exhibited a dendritic morphology while keratocytes cultured in FGF–2 and PDGF–BB appeared fibroblastic. Conclusions: These data suggest that keratocytes cultured in both insulin and ITS grow in culture and maintain their phenotype for at least 7 days. This growth factor may act to protect the core proteins of proteoglycans from degradation and thereby increase their accumulation.

Keywords: cornea: stroma and keratocytes • proteoglycans/glycosaminoglycans • wound healing