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[Culture of human keratocytes. Influence of culture conditions and ultrastructural aspects].

BACKGROUND
To investigate the influence of fetal calf serum (FCS) and fibroblast growth factor (FGF) on human keratocyte growth in vitro and cell differentiation, and to describe cultured human keratocyte ultra-structure.


METHODS
Human keratocytes were cultured in TC 199/Ham F12 media, supplemented or not with 10% FCS, aFGF, and bFGF. Keratocyte growth was studied. Cultured keratocytes were analyzed by means of immunochemistry and transmission electron microscopy.


RESULTS
Without fetal calf serum, cell population doubling occurred after 7 days of culture and no alpha smooth muscle-actin cell expression was observed. With serum, cell population increased by 1 log after 7 days of culture and all of the cells were alpha SM-actin + bFGF or aFGF-addition to the serum-containing medium resulted in a dramatic decrease in this alpha SM-actin expression. Nuclei were found to be oval and regular in cross-sections, and round and indented in frontal sections. Numerous cytoplasmic organelles were observed, as were cell expansions, gap junctions, omega-shaped structures, and fenestrations. Cultured keratocytes synthesized collagen fibers and filaments.


CONCLUSION
Fetal calf serum allows human keratocytes to grow with a myofibroblast cell phenotype, whereas addition of FGF results in a fibroblast cell phenotype. Ultrastructure of cultured keratocyte is similar to that observed in situ.