[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.