124 Pig oesophageal epithelial cells form a stratified epithelium on decellularized scaffolds in-vitro

Introduction Therapeutic options for long-gap oesophageal atresia are characterized by poor outcomes with impact on the quality of life. Tissue Engineered (TE) oesophagus could help to fill this gap. Epithelization is a critical factor to avoid long-term complication as strictures. Therefore, for a TE strategy, is crucial to create and maintain a functional epithelium. The aim of our study was to test the capacity of pig oesophageal epithelial cells (PigOEC) to engraft in oesophagus decellularized scaffolds in an in-vitro model. Methods Rat oesophagi were decellularized to prepare scaffolds using a modified detergent-enzymatic treatment protocol. PigOEC were isolated via mechanic-enzymatic dissociation from full-thickness oesophageal samples and plated over lethally-irradiated 3T3 feeder cells. PigOEC were expanded, characterized, and seeded on the inner surface of slit-open scaffolds. Cells were evaluated weekly up to 28 days. Viability was determined by colorimetric assay for cell metabolic activity (MTT assay) and samples were also processed for histological analyses. Results PigOEC were expanded long-term in culture where they showed features comparable to other mammalian epithelial cells. Seeded PigOEC successfully adhered to and covered the entire surface of the scaffolds; they were proliferating and formed a multi-layered epithelium expressing integrin α6 (CD49f) and cytokeratin (CK) 5/14 only in the basal compartment. Discussion Epithelial cells are crucial to restore the oesophageal functional barrier. We have demonstrated in vitro growth capacity of PigOEC that could differentiate and partially stratify upon seeding on decellularized scaffolds. The next steps will be testing full stratification capacity and survival of cultivated PigOEC in an in vivo model. These results support the use of PigOEC to construct a TE oesophagus for a pre-clinical study.