Esophagus tissue engineering: in vitro generation of esophageal epithelial cell sheets and viability on scaffold.

PURPOSE: Management of long gap esophageal atresia poses challenges. The surgical techniques for esophageal replacement are associated with complications and high morbidity. The aim of this study was to develop protocols to obtain single layer sheets of esophageal epithelial cells (EECs) and to investigate their survival on collagen scaffolds.

METHODS: Esophageal epithelial cells were sourced from adult Sprague-Dawley rats. Briefly, the esophagus was treated with dispase to separate the epithelial layer and further trypsined to obtained EEC. The esophageal epithelial cells were cultured in vitro and seeded on to new generation of 3-dimensional collagen scaffolds.

RESULTS: Esophageal epithelial cells organized after 48 hours in culture and formed clusters after 72 to 96 hours. Organization of the EEC was completed after 7 days in culture and characteristic sheets of EEC with the histologic morphology of mature esophageal epithelium were obtained after 14 days of culture. Immunohistochemistry demonstrated pure EEC culture using cytokeratin (CK-14) markers. The esophageal epithelial cells transferred on to collagen polymers demonstrated excellent viability after 8 weeks of in vitro culture.

CONCLUSION: Successful protocols for EEC isolation and proliferation have been established. The engineering of sheets of EEC and the viability of EEC on collagen scaffolds for 8 weeks in vitro, which are prerequisites for esophagus tissue engineering, was demonstrated.