Parameters Involved in Adhesion of Corneal Endothelial Cells Cultured In Vitro

Abstract

Abstract: : Purpose: To investigate the role of factors involved in cellular adhesion on the proliferation of corneal endothelial cells in vitro. Methods: Human, bovine, porcine, rabbit corneal endothelial cells were cultured in different types of substrates: poli-lysin, fibronectine, matrigel, collagen type I, extracellular matrix from corneal endothelial bovine cell and plastic. Different kinds of growth factors and medium components were tested on cell cultures. Cultured cells growth and viability were evaluated by phase contrast microscopy, growth kinetics and by ³H-thymidine incorporation. The expression of adhesion proteins, vimentine and pan-cytokeratine, was tested with immunohistochemistry on both sections of fresh corneas and on cell cultures. Results: Cell cultures were compared. In non adherent culture conditions no growth was observed and cells died within few days from primary culture. Otherwise, when cells allowed to adhere to the substrate, they could grow and reach confluence within 15 days. Bovine and rabbit corneal endothelial cells grew on poli-lysin, matrigel, collagen type I and extracellular matrix from corneal endothelial bovine cells. No significative growth of these cells was observed on fibronectine and plastic. The best results for the growth of porcine and human corneal endothelial cells were observed on matrigel and extracellular matrix from corneal endothelial bovine cells. No significative growth was observed on the others substrates tested. When medium was additionated with FGF-2, EGF, ascorbic acid and cheratocytes conditioned medium cell growth was faster. Chondroitin sulphate influenced positively cells adherence on substrates. Vimentine and pan-cytocheratine, as evidenced by immunocytochemistry, resulted expressed on every endothelium sections obtained by fresh corneas. These markers were also positive on proliferating cell cultures, but not in quiescent cells. No vimentine and pan-cytocheratine were expressed on cell cultured as non adherent. Conclusions: These results suggest that adhesion molecules may play an important role on proliferation of human corneal endothelial cells in vitro, suggesting that these cells require an appropriate substrate to start the activation of genes leading to proliferation.

Keywords: cornea: endothelium • cell adhesions/cell junctions • extracellular matrix