Fabrication of cultivated human corneal endothelial cell sheets using a temperature– responsive polymer substrate.

Abstract

Abstract: : Purpose: To fabricate cultivated human corneal endothelial cell (HCEC) sheets for clinical use, and determine if they have similar characteristics to the endothelium in vivo. Methods: HCECs cultured on a temperature–responsive polymer (PIPAAm) were detached and investigated by light and transmission/scanning electron microscopy. Extracellular matrix was examined by immunofluorescence of collagen type IV and fibronectin. Cell–cell junction formation was studied by immunostaining for the tight junction protein ZO–1. Influences of harvesting methods were examined by immunoblotting for ZO–1. Results: After HCEC sheets were partly released and removed from the PIPAAm culture surfaces by reducing the temperature to 20 C, no remnant matrix material was found adhered to the surfaces. Some aspects of HCEC morphology resembled endothelial cells in vivo, although differences did exist. With time, the accumulation of extracellular matrix underlying the sheet increased. Positive staining for ZO–1 was seen in the intercellular area. Immunoblotting indicated that enzymatic harvest degraded ZO–1, whereas low temperature harvest did not. Conclusions: We fabricated HCEC sheets using a temperature–responsive culture system. The results indicate highly promising clinical capabilities for our bioengineered tissue constructs for surgical intervention to treat corneal endothelial diseases.

Keywords: cornea: endothelium • transplantation • extracellular matrix