Human Anterior Lens Capsule as Carrier Matrix for Cultivated Human Corneal Endothelial Cells

Abstract

To evaluate the potential of human anterior lens capsule (HALC) as a carrier matrix for cultivating and transplanting human corneal endothelial cells (HCECs). HALCs obtained from 12 patients during cataract surgery were exposed to enzyme digestion to dissolve the lens epithelium and were plated with the epithelial side up in 6-well plates. HCECs were harvested from human donor eyes and seeded on HALCs. Cell morphology was assessed with a phase-contrast microscope after 6 hours of incubation and at days 1, 3, 5, and 7. The number of HCECs counted at each capsule was pooled, and the density was expressed as cells per square millimeter after 7 days in culture. Live/Dead viability/cytotoxicity assay was also performed and analyzed by fluorescence microscopy after 7 days of incubation during confluence. Expression of zonula occludens-1, connexin-43, Na/K-adenosine triphosphatase, and cytokeratin-3 (AE5) were investigated by immunohistochemistry. Seven days after seeding on HALCs, the HCECs were grown to confluence and formed a continuous viable monolayer with a mean cell density of 3012 +/- 109 cells per square millimeter, which mimics native corneal endothelial cells. Immunohistochemistry analysis demonstrated strongly positive staining for AE5, zonula occludens-1, connexin-43, and Na/K-adenosine triphosphatase. Cell density and morphology of HCECs on HALCs were similar to those of healthy corneas. Phenotypical properties of HCECs on HALCs imply that the HCEC sheets are capable of maintaining intact barrier and ionic pump functions in vitro. HALCs might, therefore, be recommended as a potential scaffold for ex vivo expansion of HCECs, possibly providing an autologous biologic substrate for therapy of isolated corneal endothelial diseases.