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 3 weeks at 37°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°C. In vitro characteristics of theHCECsheets 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.
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