Objective: To investigate whether the bioengineered human corneal endothelial cell (HCEC) monolayers harvested from thermoresponsive culture supports could be used as biological tissue equivalents. Methods: Untransformed adult HCECs derived from eye bank corneas were cultivated on a thermoresponsive poly-N-isopropylacrylamide-grafted surface for 3weeks at 37 degrees C. Confluent cell cultures with a phenotype and cell density similar to HCECs in vivo were detached as a laminated sheet by lowering the culture temperature to 20 degrees C. In vitro characteristics of the HCEC sheets were determined evaluating their viability and by scanning electron microscopy, immunohistochemistry, and histological studies. Results: After separation from culture surfaces via a thermal stimulus, the HCEC sheets remained viable. Polygonal cell morphology and multiple cellular interconnections were observed throughout the HCEC sheets. Immunolocalization of zonula occludens-1 and Na+, K+ adenosine triphosphatase (ATPase) indicated the formation of tight junctions and the distribution of ionic pumps at the cell boundary. In addition, we ascertained that cultured HCECs have a monolayered architecture that mimics native corneal endothelium. Conclusion: These data suggest that a well-organized and functional HCEC monolayer can feasibly be used as tissue equivalents for replacing compromised endothelium. Clinical Relevance: Bioengineered human corneal endothelium fabricated from thermoresponsive supports can potentially offer a new therapeutic strategy for corneal endothelial cell loss.
