Culture of ovine esophageal epithelial cells and in vitro esophagus tissue engineering.

摘要

BACKGROUND: Esophagus replacement presents major surgical challenges both in the pediatric and in adult patients since the various surgical techniques presently employed are associated with complications and high morbidity. AIM: The aim of this study was to establish protocols for isolation and culture of ovine esophageal epithelial cells (OEEC) and to investigate their viability on collagen scaffolds for in vitro tissue engineering. METHODS: OEEC were sourced from adult Austrian mountain sheep. Briefly, the esophagus was dissected, treated with dispase to separate the epithelial layer, and further subjected to a modified explants technique to isolate OEEC. The OEEC were cultured in vitro and seeded on to unidirectional two-dimensional and three-dimensional collagen scaffolds. RESULTS: Successful protocol was established for OEEC isolation and culture. OEEC exhibited organization and differentiation after 7 days in culture, which was complete after 18 days with the formation of a single layer sheet of differentiated cells exhibiting morphology of mature esophageal epithelium. OEEC seeded on two-dimensional collagen scaffolds demonstrated viability up to 6 weeks of in vitro culture with single layer epithelium formation after 3 weeks confirmed using pan-Cytokeratin markers. OEEC on three-dimensional scaffolds were viable for 6 weeks but did not form an epithelium sheet. CONCLUSION: Protocols for OEEC isolation were developed and established from adult ovine esophageal tissue. The generation of sheets of esophageal epithelium in culture and the viability of OEEC on collagen scaffolds for 6 weeks in vitro was observed. The prerequisite for esophagus tissue engineering, which is the ability to form epithelium when seeded on collagen scaffolds, was demonstrated.