Influence of fetal calf serum, fibroblast growth factors, and hepatocyte growth factor on three-dimensional cultures of human keratocytes in collagen gel matrix

Background: We set out to evaluate the influence of fetal calf serum, acidic fibroblast growth factor (aFGF), basic fibroblast growth factor (bFGF), and hepatocyte growth factor (HGF) on three-dimensional cultures of human keratocytes in type I collagen gel matrix. Methods: Polymerized gels were cultured at 37 degrees C for 35 days. Gel contraction and integrated optical density were assessed 3 times weekly for 5 weeks using an image analysis system. Gels were studied at the end of the culture period by means of transmission electron microscopy (TEM) and immunochemistry. Results: Serum significantly increased gel contraction and decreased gel optical transmittance. Keratocyte density was significantly increased by serum and HGF. In TEM, collagen density was higher with serum-supplemented media than with serum-free media, and higher with HGF-supplemented media than with HGF-free media. Immunoperoxidase staining of keratocyte-populated gels showed positive staining for vimentin, connexin 43, and type I, type V, and type VI human collagen, whereas no expression of desmin, alpha smooth muscle actin, and type IV collagen was observed. Expression of type I collagen was significantly increased by aFGF and HGF, expression of type VI collagen by serum and bFGF. Conclusion: Serum and HGF improve ultrastructural and immunochemical features of human keratocyte-populated collagen gels.