A human corneal endothelium equivalent constructed with acellular porcine corneal matrix

BACKGROUND & OBJECTIVES Artificial corneal endothelium equivalents can not only be used as in vitro model for biomedical research including toxicological screening of drugs and investigation of pathological corneal endothelium conditions, but also as potential sources of grafts for corneal keratoplasty. This study was aimed to demonstrate the feasibility of constructing human corneal endothelium equivalents using human corneal endothelial cells and acellular porcine corneal matrix. METHODS Porcine corneas were decellularized with sodium dodecyl sulphate (SDS) solution. Human corneal endothelial cells B4G12 were cultured with leaching liquid extracted from the acellular porcine corneal matrix, and then cell proliferative ability was evaluated by MTT assay. B4G12 cells were transplanted to a rat corneal endothelial deficiency model to analyze their in vivo bio-safety and pump function, and then seeded and cultured on acellular porcine corneal matrix for two wk. Corneal endothelium equivalents were analyzed using HE staining, trypan blue and alizarin red S co-staining, immunofluorescence and corneal swelling assay. RESULTS The leaching liquid from acellular porcine corneal matrix had little influence on the proliferation ability of B4G12 cells. Animal transplantation of B4G12 cells showed that these cells had similar function to the native cells without causing a detectable immunological reaction and neoplasm in vivo. These formed a monolayer covering the surface of the acellular porcine corneal matrix. Trypan blue and alizarin red S co-staining showed that B4G12 cells were alive after two wk in organ culture and cell boundaries were clearly delineated. Proper localizations of ZO-1 and Na+/K+ ATPase were detected by immunofluorescence assay. Functional experiments were conducted to show that the Na+/K+ ATPase inhibitor ouabain could block the ionic-pumping function of this protein, leading to persistent swelling of 51.7 per cent as compared to the control. INTERPRETATION & CONCLUSIONS Our findings showed that B4G12 cells served as a good model for native corneal endothelial cells in vivo. Corneal endothelium equivalents had properties similar to those of native corneal endothelium and could serve as a good model for in vitro study of human corneal endothelium.