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T. Ide, K. Nishida, M. Yamato, T. Sumide, A. Kikuchi, T. Okano, Y. Tano; Characteristics of Cultured Human Corneal Endothelial Cells Changes Depending on Substrates . Invest. Ophthalmol. Vis. Sci. 2005;46(13):4518.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: For clinical use we are now try to make cultivated human corneal endothelial cell (HCEC) sheets. An important step is to find appropriate substrates for cell culture. Here we investigate the characteristics of HCECs seeded on different substrates. Methods: We performed alizarin red S staining of HCECs cultured on (i) plastic dishes, (ii) polyethylene terephthalate (PET) insert membranes, and (iii) mixed cellulose esters (HA) insert membranes. To evaluate barrier function we used FITC–dextran (for non–ionic flow) and transmonolayer electrical resistance (TER; for ionic flow). The expression of tight junction–related proteins was investigated by immunoblotting for occludin and claudin–1. All experiments were performed either with or without ouabain (a specific Na–K–ATPase inhibitor) or cytochalasin D (an inhibitor of F–actin polymerization). Results: Only HCECs on HA were positive for alizarin red S. Differences in culture substrates and treatment with ouabain or cytochalasin D did not affect the barrier function values measured by TER. HCECs on PET formed a strong barrier against the passage of FITC–dextran with or without ouabain or cytochalasin D, whereas HCECs on HA formed a weaker barrier. When ouabain or cytochalasin D were added to HCECs on HA the barrier function was reduced in a dose–dependent manner. The strengths of the occludin and claudin–1 expressions correlated with the strength of the barrier function obtained from FITC–dextran assays. Conclusions: The positive alizarin red S staining and barrier kinetics in response to ouabain and cytochalasin D that are seen in HCECs cultured on HA indicate similarities to HCECs in vivo. Thus, a HA culture system might be desirable for the fabrication of functional HCEC sheets.
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