Comparative study of four culture methods to engineer murine corneal epithelial sheet

• Aim: To investigate the roles of feeder cells in stratification of murine corneal epithelial cells and build an ideal method to engineer stratified epithelial sheet. • Methods: Using contact feeder culture, separated feeder culture, compound feeder culture and culture without feeder cells by Air-lifting method in Transwell chamber culture system, tissue engineered corneal epithelium was reconstructed. Corneal sheets were stained with hematoxylin and eosin (HE) for histological observation. The expression of p63 and keratin 19 (K19) and involucrin (IVL) was investigated by immunocytochemistry analysis. • Results: Stratification was limited to three to four layers in the contact feeder group, whereas separate feeder sheets were slightly more stratified. The compound feeder group produced a stratified epithelium with five to seven layers of cells. The group without 3T3 feeder cells formed only two to three layers of cells. Immunostaining images in the compound feeder group showed expression of progenitor markers p63 and K19 in the basal and suprabasal layer, as well as differentiation marker involucrin in all layers. • Conclusion: The remarkable stratification as well as the limbal phenotype makes the compound feeder system a candidate tool for cultivating transplantable epithelial sheets.